Phosphate replacement mrna cap analogs

ABSTRACT

The present disclosure relates to cap analogs, which can result in high levels of capping efficiency and transcription and improved translation efficiencies. The present disclosure also relates to methods useful for preparing cap analogs and using mRNA species containing such analogs, as well as kits containing the novel cap analogs.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/768,199, filed Apr. 13, 2018, which is a U.S. National Phaseapplication, filed under 35 U.S.C. § 371, of International ApplicationNo. PCT/US2016/057384, filed Oct. 17, 2016, which claims priority to,and the benefit of, U.S. Provisional Application No. 62/242,845, filedOct. 16, 2015, the entire contents of which are incorporated herein byreference in their entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “MRNA008C03SL.txt”, which wascreated on Jul. 9, 2018, and is 764 bytes in size, are incorporatedherein by reference in its entirety.

BACKGROUND

Expression of the genetic information coded by a sequence of nucleotidesin deoxyribonucleic acid (DNA) requires a biosynthesis of acomplementary messenger ribonucleic acid (mRNA). This transcriptionevent, which takes place in the nucleus of eukaryotic cells, is followedby translocation of the mRNA into the cytoplasm, where it is loaded intoribosomes by a complex and highly regulated process. Here the nucleotidesequence, presented as a series of three-nucleotide codons is translatedinto a corresponding sequence of amino acids ultimately producing theprotein corresponding to the original genetic code.

Exogenous mRNA introduced to the cytoplasm can be in principle acceptedby the ribosomal machinery (see, e.g., Warren et al., Highly EfficientReprogramming to Pluripotency and Directed Differentiation of HumanCells with Synthetic Modified mRNA, Cell Stem Cell (2010)). If the mRNAcodes for an excreted protein, the modified or exogenous mRNA can directthe body's cellular machinery to produce a protein of interest, fromnative proteins to antibodies and other entirely novel proteinconstructs that can have therapeutic activity inside and outside ofcells.

There are difficulties with prior methodologies for effecting proteinexpression. There is a need in the art for biological modalities toaddress the modulation of intracellular translation of polynucleotides.

SUMMARY

The present disclosure provides mRNA cap analogs and methods of makingand using them. The present disclosure also provides mRNA containing thecap analogs.

In one aspect, the present disclosure features a compound of formula (I)below or a stereoisomer, tautomer or salt thereof:

In formula (I) above,

ring B₁ is a modified Guanine;

ring B₂ is a nucleobase or a modified nucleobase;

X₂ is O, S(O)_(p), NR₂₄ or CR₂₅R₂₆ in which p is 0, 1, or 2;

Y₂ is —(CR₄₀R₄₁)_(u)-Q₀-(CR₄₂R₄₃)_(v)—, in which Q₀ is a bond, O,S(O)_(r), NR₄₄, or CR₄₅R₄₆, r is 0, 1, or 2, and each of u and vindependently is 1, 2, 3 or 4;

R₂ is halo, LNA, or OR₃;

R₃ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl and R₃, when beingC₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, is optionally substitutedwith one or more of halo, OH and C₁-C₆ alkoxyl that is optionallysubstituted with one or more OH or OC(O)—C₁-C₆ alkyl;

each of R₂₀, R₂₁, R₂₂, and R₂₃ independently is -Q₃-T₃, in which Q₃ is abond or C₁-C₃ alkyl linker optionally substituted with one or more ofhalo, cyano, OH and C₁-C₆ alkoxy, and T₃ is H, halo, OH, NH₂, cyano,NO₂, N₃, R_(S3), or OR_(S3), in which R_(S3) is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, NHC(O)—C₁-C₆alkyl, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, 4 to 12-memberedheterocycloalkyl, or 5- or 6-membered heteroaryl, and R_(S3) isoptionally substituted with one or more substituents selected from thegroup consisting of halo, OH, oxo, C₁-C₆ alkyl, COOH, C(O)O—C₁-C₆ alkyl,cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, and 5-or 6-membered heteroaryl;

each of R₂₄, R₂₅, and R₂₆ independently is H or C₁-C₆ alkyl;

each of R₂₇ and R₂₈ independently is H or OR₂₉; or R₂₇ and R₂₈ togetherform O—R₃₀—O;

each R₂₉ independently is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl and R₂₉, when being C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl, is optionally substituted with one or more of halo, OH andC₁-C₆ alkoxyl that is optionally substituted with one or more OH orOC(O)—C₁-C₆ alkyl;

R₃₀ is C₁-C₆ alkylene optionally substituted with one or more of halo,OH and C₁-C₆ alkoxyl;

each of R₄₀, R₄₁, R₄₂, and R₄₃ independently is H, halo, OH, cyano, N₃,OP(O)R₄₇R₄₈, or C₁-C₆ alkyl optionally substituted with one or moreOP(O)R₄₇R₄₈, or one R₄₁ and one R₄₃, together with the carbon atoms towhich they are attached and Q₀, form C₄-C₁₀ cycloalkyl, 4- to14-membered heterocycloalkyl, C₆-C₁₀ aryl, or 5- to 14-memberedheteroaryl, and each of the cycloalkyl, heterocycloalkyl, phenyl, or 5-to 6-membered heteroaryl is optionally substituted with one or more ofOH, halo, cyano, N₃, oxo, OP(O)R₄₇R₄₈, C₁-C₆ alkyl, C₁-C₆ haloalkyl,COOH, C(O)O—C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₁-C₆ haloalkoxyl, amino,mono-C₁-C₆ alkylamino, and di-C₁-C₆ alkylamino;

R₄₄ is H, C₁-C₆ alkyl, or an amine protecting group;

each of R₄₅ and R₄₆ independently is H, OP(O)R₄₇R₄₈, or C₁-C₆ alkyloptionally substituted with one or more OP(O)R₄₇R₄₈, and

each of R₄₇ and R₄₈, independently is H, halo, C₁-C₆ alkyl, OH, SH, SeH,or BH₃ ⁻.

The present disclosure also provides an RNA molecule (e.g., mRNA) whose5′ end contains a compound of formula (I).

Also provided herein is a kit for capping an RNA transcript. The kitincludes a compound of formula (I) and an RNA polymerase. The kit mayalso include one or more of nucleotides, ribonuclease inhibitor, anenzyme buffer, and a nucleotide buffer.

In yet another aspect, the present disclosure provides methods ofsynthesizing the compound of formula (I).

In still another aspect, the present disclosure provides methods ofsynthesizing an RNA molecule (e.g., mRNA) in vitro. The method caninclude reacting unmodified or modified ATP, unmodified or modified CTP,unmodified or modified UTP, unmodified or modified GTP, a compound offormula (I) or a stereoisomer, tautomer or salt thereof, and apolynucleotide template; in the presence an RNA polymerase; under acondition conducive to transcription by the RNA polymerase of thepolynucleotide template into one or more RNA copies; whereby at leastsome of the RNA copies incorporate the compound of formula (I) or astereoisomer, tautomer or salt thereof to make an RNA molecule (e.g.,mRNA).

In yet another aspect, the present disclosure provides a compound (e.g.,a cap analog) or a polynucleotide containing the cap analog having animproved eIF4E binding affinity, enhanced resistance to degradation, orboth, as compared to, e.g., natural mRNA caps and natural mRNAs.

Further, the compounds or methods described herein can be used forresearch (e.g., studying interaction of in vitro RNA transcript withcertain enzymes) and other non-therapeutic purposes.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting. In the case ofconflict between the chemical structures and names of the compoundsdisclosed herein, the chemical structures will control.

Other features and advantages of the disclosure will be apparent fromthe following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plot of normalized relative fluorescence units (RFU) vs. theconcentrations of the cap analogs tested from a cell free translationassay.

FIG. 2 is a histogram of hEPO levels measured after 3 hours of a cellfree translation assay using mRNAs carrying different cap analogs,comparing the hEPO levels normalized for % capping obtained using anmRNA carrying Compound 7 to that of an mRNA carrying a triphosphate cap(“Standard” in FIG. 2, with a chemical structure of

The Compound 7-cap-carrying mRNA shows superior expression (comparableto that of ARCA1) in a HeLa-derived cell free system, when compared tothe triphosphate analog and/or Capt. In this Figure, “mod mRNA” refersto a modified mRNA comprising N1-methyl pseudouridine, which replaceseach uridine in the RNA sequence.

DETAILED DESCRIPTION

The present disclosure provides novel mRNA cap analogs, syntheticmethods for making these cap analogs, and uses thereof. The presentdisclosure also provides new RNA molecules (e.g., mRNAs) incorporatingthe cap analogs disclosed herein which impart properties that areadvantageous to therapeutic development.

The mRNA consists of an open reading frame (ORF) flanked by the 5′- and3′-untranslated region (5′UTR, 3′UTR), a poly-adenosine monophosphatetail (polyA) and an inverted N7-methylguanosine containing capstructure. It is both chemically and enzymatically less stable than thecorresponding DNA, hence the protein production subsequent to theribosomal recruitment of the mRNA is temporary. In addition, the mRNAmust be present in a so-called “closed loop” conformation for productionof the target protein. While part of the active closed-loopconformation, the mRNA makes contact with the ribosomal machinerythrough the cap that binds to the eukaryotic initiation factor 4E(eIF4E) and the polyA tail attached through the polyA-binding protein(PABP). The eIF4E and PABP are connected through a skeletal proteineIF4G closing the active loop. Disruption of the mRNA circularized formleads to cessation of protein production and eventually enzymaticdegradation of the mRNA itself chiefly by action of the de-cappingenzyme system DCP1/2 and or through a poly-A ribonuclease (PARN)mediated de-adenylation. See, e.g., Richard J. Jackson et al., “Themechanism of eukaryotic translation initiation and principles of itsregulation”, Molecular Cell Biology, vol. 110, 113-127, 2010.

The cap-structure is a crucial feature of all eukaryotic mRNAs. It isrecognized by the ribosomal complex through the eukaryotic initiationfactor 4E (eIF4E). mRNAs lacking the 5′-cap terminus are not recognizedby the translational machinery and are incapable of producing the targetprotein (see, e.g., Colin Echeverria Aitken, Jon R Lorsch: “Amechanistic overview of translation initiation in eukaryotes”, NatureStructural and Molecular Biology, vol. 16, no. 6, 568-576, 2012.)

The crude messenger RNA produced during the transcription process(“primary transcript”) is terminated by a 5′-triphosphate, which isconverted to the respective 5′-diphosphate by the action of the enzymeRNA-triphosphatase. Then a guanylyl-transferase attaches the terminalinverted guanosine monophosphate to the 5′-terminus, and anN7MTase-mediated N7-methylation of the terminal, inverted guanosine,completes the capping process.

The 5′-cap structure is vulnerable to enzymatic degradation, which ispart of the regulation mechanism controlling protein expression.According to this the enzymatic system DCP1/2 performs a pyrophosphatehydrolysis between the second and the third phosphate groups of the capstructure, removing the N7-methylated guanosine diphosphate moietyleaving behind an mRNA terminated in a 5′-monophosphate group. This inturn is quite vulnerable to exonuclease cleavage and will lead to rapiddecay of the remaining oligomer. See, e.g., R. Parker, H. Song: “TheEnzymes and Control of Eukaryotic Turnover”, Nature Structural &Molecular Biology, vol. 11, 121-127, 2004.

High resolution X-ray crystallographic data of the eukaryotic initiationfactor 4E (eIF4E) co-crystallized withP1-N7-methylguanosine-P3-adenosine-5′,5′-triphosphate (N7GpppA) suggestsa close molecular interaction between the terminal purine and thetriphosphate moiety on one hand and the receptor surface on the other.See, e.g., Koji Tomoo, et al., “Crystal structures of 7-methylguanosine5′-triphosphate (m(7)GTP)- andP(1)-7-methylguanosine-P(3)-adenosine-5′,5′-triphosphate(m(7)GpppA)-bound human full-length eukaryotic initiation factor 4E:biological importance of the C-terminal flexible region.”, Biochem. J.362(Pt 3): 539-544, 2002. The terminal guanine is sandwiched between twoaromatic side chains of TRP56 and TRP102 and this π-stacking interactionis further stabilized by two hydrogen bonds between the N7-guanine NHhydrogens and GLU103. The first two phosphate groups are interactingwith basic residues of ARG112 and ARG157 as well as LYS162 eitherdirectly or through water mediated hydrogen bonds. The third phosphategroup forms a hydrogen bond with the basic residue of ARG112. In short,the high resolution x-ray crystallographic data suggests that the boththe guanine and the triphosphate make direct contact with the proteinand contribute to the binding efficiency of capped mRNAs.

The triphosphate moiety of the eukaryotic cap structure plays animportant role in binding to the eIF4E as well as the stability of themRNA. See, e.g., Anna Niedzwiecka et al., “Biophysical Studies of eIF4ECap-binding Protein: Recognition of mRNA 50 Cap Structure and SyntheticFragments of eIF4G and 4E-BP1 Proteins.”, Journal of Molecular Biology,319, 615-635, 2002. In addition, a DCP1/2-mediated hydrolysis of thepyrophosphate bond is the chief de-capping mechanism. See, e.g., EwaGrudzien et al., “RNA: Structure, Metabolism, and Catalysis:Differential Inhibition of mRNA Degradation Pathways by Novel CapAnalogs.”, Journal of Biological Chemistry, 281:1857-1867, 2006.Accordingly, the present disclosure is based, at least in part, on theassumption that replacement of the central phosphate with hydrophilicgroups will be capable of maintaining the affinity to the bindingpocket. Without wishing to be bound by theory, replacement of thisphosphate with non-phosphate bridges could decrease the overallelectrophilicity of the pyrophosphate grouping present in triphosphates,and since de-capping requires a nucleophilic attack of an enzyme-guidedwater molecule on the γ-phosphate, this in turn will result in capstructures resistant to enzymatic hydrolysis. The internucleosidedistance can be tuned by the choice of appropriately sized moietyreplacing the central triphosphate, such as glycols (e.g., diethylene-or triethylene-glycols). In addition to the distance, the overallspecial orientation of the two nucleosides can be altered by inclusionof cyclic structures such as cyclohexane-1,3-diol. Presence ofhydrophilic groups such as that of a sulfoxide (SO), sulfone (SO₂) oreven a phosphate can facilitate interaction with the polar groups liningthe eIF4E binding pocket, while maintaining chemical and enzymaticstability. These chemical modifications will have an impact on cap'sbinding affinity to the eIF4E. In addition to altered binding behavior,these chemical modifications will affect the affinity of these capstowards the DCP1/2 enzyme system, and potentially improve stability ofthe respective mRNA. This will allow for development of novel distinctSAR for these new cap structures for eIF4E-cap protein and lead tomessenger RNA caps with improved eIF4E binding, and enhanced resistanceto degradation, which in turn can result in increased rate oftranslation, extended stability of the “closed-loop” conformation andenhanced production of target proteins of therapeutic value.

In one aspect, the present disclosure provides a compound (e.g., a capanalog) of formula (I) below or a stereoisomer, tautomer or saltthereof:

In formula (I) above,

ring B₁ is a modified Guanine;

ring B₂ is a nucleobase or a modified nucleobase;

X₂ is O, S(O)_(p), NR₂₄ or CR₂₅R₂₆ in which p is 0, 1, or 2;

Y₂ is —(CR₄₀R₄₁)_(u)-Q₀-(CR₄₂R₄₃)_(v)—, in which Q₀ is a bond, O,S(O)_(r), NR₄₄, or CR₄₅R₄₆, r is 0, 1, or 2, and each of u and vindependently is 1, 2, 3 or 4;

R₂ is halo, LNA, or OR₃;

R₃ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl and R₃, when beingC₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, is optionally substitutedwith one or more of halo, OH and C₁-C₆ alkoxyl that is optionallysubstituted with one or more OH or OC(O)—C₁-C₆ alkyl;

each of R₂₀, R₂₁, R₂₂, and R₂₃ independently is -Q₃-T₃, in which Q₃ is abond or C₁-C₃ alkyl linker optionally substituted with one or more ofhalo, cyano, OH and C₁-C₆ alkoxy, and T₃ is H, halo, OH, NH₂, cyano,NO₂, N₃, R_(S3), or OR_(S3), in which R_(S3) is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, NHC(O)—C₁-C₆alkyl, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, 4 to 12-memberedheterocycloalkyl, or 5- or 6-membered heteroaryl, and R_(S3) isoptionally substituted with one or more substituents selected from thegroup consisting of halo, OH, oxo, C₁-C₆ alkyl, COOH, C(O)O—C₁-C₆ alkyl,cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, and 5-or 6-membered heteroaryl;

each of R₂₄, R₂₅, and R₂₆ independently is H or C₁-C₆ alkyl;

each of R₂₇ and R₂₈ independently is H or OR₂₉; or R₂₇ and R₂₈ togetherform O—R₃₀—O;

each R₂₉ independently is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl and R₂₉, when being C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl, is optionally substituted with one or more of halo, OH andC₁-C₆ alkoxyl that is optionally substituted with one or more OH orOC(O)—C₁-C₆ alkyl;

R₃₀ is C₁-C₆ alkylene optionally substituted with one or more of halo,OH and C₁-C₆ alkoxyl;

each of R₄₀, R₄₁, R₄₂, and R₄₃ independently is H, halo, OH, cyano, N₃,OP(O)R₄₇R₄₈, or C₁-C₆ alkyl optionally substituted with one or moreOP(O)R₄₇R₄₈, or one R₄₁ and one R₄₃, together with the carbon atoms towhich they are attached and Q₀, form C₄-C₁₀ cycloalkyl, 4- to14-membered heterocycloalkyl, C₆-C₁₀ aryl, or 5- to 14-memberedheteroaryl, and each of the cycloalkyl, heterocycloalkyl, phenyl, or 5-to 6-membered heteroaryl is optionally substituted with one or more ofOH, halo, cyano, N₃, oxo, OP(O)R₄₇R₄₈, C₁-C₆ alkyl, C₁-C₆ haloalkyl,COOH, C(O)O—C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₁-C₆ haloalkoxyl, amino,mono-C₁-C₆ alkylamino, and di-C₁-C₆ alkylamino;

R₄₄ is H, C₁-C₆ alkyl, or an amine protecting group;

each of R₄₅ and R₄₆ independently is H, OP(O)R₄₇R₄₈, or C₁-C₆ alkyloptionally substituted with one or more OP(O)R₄₇R₄₈, and

each of R₄₇ and R₄₈, independently is H, halo, C₁-C₆ alkyl, OH, SH, SeH,or BH₃.

The compound of formula (I) or a stereoisomer, tautomer or salt thereofcan have one or more of the following features when applicable.

For example, R₂ is halo (e.g., fluorine, chlorine, bromine, and iodine).

For example, R₂ is fluorine.

For example, R₂ is LNA.

For example, R₂ is OR₃.

For example, R₃ is H.

For example, R₃ is C₁-C₃ alkyl, e.g., methyl.

For example, R₃ is C₁-C₃ alkyl substituted with one or more of C₁-C₆alkoxyl that is optionally substituted with one or more OH orOC(O)—C₁-C₆ alkyl.

For example, R₃ is CH₂CH₂OCH₃.

For example, R₃ is CH(OCH₂CH₂OH)₂.

For example, R₃ is CH(OCH₂CH₂OCOCH₃)₂.

For example, R₃ is unsubstituted or substituted C₂-C₆ alkenyl, e.g.,propen-3-yl.

For example, R₃ is unsubstituted or substituted C₂-C₆ alkynyl, e.g.,propyn-3-yl.

For example, ring B₁ is

in which

R₁ is C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, each of which isoptionally substituted with one or more substituents selected from thegroup consisting of C₆-C₁₀ aryl, C₆-C₁₀ aryloxyl, 5- to 10-memberedheteroaryl, and 5- to 10-membered heteroaryloxyl, each being optionallysubstituted with one or more of halo and cyano;

each of R_(a) and R_(b) independently is H or C₁-C₆ alkyl; and

R_(c) is H, NH₂, or C₁-C₆ alkyl; or R_(c) and one of R_(a) and R_(b),together with the two nitrogen atoms to which they attach and the carbonatom connecting the two nitrogen atoms form a 5- or 6-memberedheterocycle which is optionally substituted with one or more of OH,halo, C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl,

a stereoisomer, tautomer or salt thereof.

For example, ring B₁ is

in which each of R_(a), R_(b) and R_(c) independently is H or C₁-C₆alkyl.

For example, ring B₁ is

in which each of R_(a), R_(b) and R_(c) independently is H or C₁-C₆alkyl.

For example, ring B₁ is

in which each of R_(a), R_(b) and R_(c) independently is H or C₁-C₆alkyl, and R₁ is C₁-C₆ alkyl or C₂-C₆ alkenyl (e.g., propen-3-yl).

For example, each of R_(a) and R_(b) independently is H or C₁-C₃ alkyl.

For example, R_(c) is H.

For example, R_(c) is NH₂.

For example, R_(c) and one of R_(a) and R_(b), together with the twonitrogen atoms to which they attach and the carbon atom connecting thetwo nitrogen atoms form a 5- or 6-membered heterocycle which isoptionally substituted with one or more of OH, halo, C₁-C₆ alkyl, C₂-C₆alkenyl, and C₂-C₆ alkynyl. For example, the other of R_(a) and R_(b)that does not form the heterocycle is absent, H, or C₁-C₆ alkyl.

For example, ring B₁ is

in which each of R_(g) and R_(h) independently is H or C₁-C₃ alkyl.

For example, R_(g) is H or methyl.

For example, R_(b) is H or methyl.

For example, R₁ is C₁-C₃ alkyl.

For example, R₁ is methyl.

For example, R₁ is ethyl substituted with phenoxyl that is substitutedwith one or more of halo and cyano.

For example, R₁ is 4-chlorophenoxylethyl, 4-bromophenoxylethyl, or4-cyanophenoxylethyl.

For example, R₁ is C₂-C₆ alkenyl (e.g., propen-3-yl).

For example, ring B₂ is

in which

X₁ is N or N⁺(R₅);

R₅ is C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, each of which isoptionally substituted with one or more substituents selected from thegroup consisting of C₆-C₁₀ aryl, C₆-C₁₀ aryloxyl, 5- to 10-memberedheteroaryl, and 5- to 10-membered heteroaryloxyl, each being optionallysubstituted with one or more of halo and cyano;

each of R_(d) and R_(e) independently is H or C₁-C₆ alkyl; and

R_(f), when present, is H, NH₂, or C₁-C₆ alkyl; or R_(f) and one ofR_(d) and R_(e), together with the two nitrogen atoms to which theyattach and the carbon atom connecting the two nitrogen atoms form a 5-or 6-membered heterocycle which is optionally substituted with one ormore of OH, halo, C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl, or astereoisomer, tautomer or salt thereof.

For example, each of R_(d) and R_(e) independently is H or C₁-C₃ alkyl.

For example, R_(d) is H or methyl.

For example, R_(e) is H or methyl.

For example, R_(f), when present, is H.

For example, R_(f), when present, is NH₂.

For example, R_(f), when present, is C₁-C₆ alkyl.

For example, R_(f) and one of R_(d) and R_(e), together with the twonitrogen atoms to which they attach and the carbon atom connecting thetwo nitrogen atoms form a 5- or 6-membered heterocycle which isoptionally substituted with one or more of OH, halo, C₁-C₆ alkyl, C₂-C₆alkenyl, and C₂-C₆ alkynyl. For example, the other of R_(d) and R_(e)that does not form the heterocycle is absent, H, or C₁-C₆ alkyl.

For example, ring B₂ is

in which each of R_(g) and R_(h) independently is H or C₁-C₃ alkyl. Forexample, R_(g) is H or methyl. For example, R_(h) is H or methyl.

For example, X₁ is N.

For example, X₁ is N⁺(R₅).

For example, R₅ is methyl.

For example, R₅ is ethyl substituted with phenoxyl that is substitutedwith one or more of halo and cyano.

For example, R₅ is 4-chlorophenoxylethyl, 4-bromophenoxylethyl, or4-cyanophenoxylethyl.

For example, X₂ is O.

For example, X₂ is S, SO, or SO₂.

For example, X₂ is NR₂₄.

For example, X₂ is CR₂₅R₂₆.

For example, R₂₄ is H.

For example, R₂₄ is straight chain C₁-C₆ or branched C₃-C₆ alkyl,including but not limited to, methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl and n-hexyl.

For example, R₂₅ is H.

For example, R₂₅ is straight chain C₁-C₆ or branched C₃-C₆ alkyl,including but not limited to, methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl and n-hexyl.

For example, R₂₆ is H.

For example, R₂₆ is straight chain C₁-C₆ or branched C₃-C₆ alkyl,including but not limited to, methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl and n-hexyl.

For example, each of R₂₅ and R₂₆ is H.

For example, R₂₇ is H.

For example, R₂₈ is H.

For example, R₂₇ is OH.

For example, R₂₈ is OH.

For example, both R₂₇ and R₂₈ are OH.

For example, R₂₇ is OR₂₉.

For example, R₂₈ is OR₂₉.

For example, both R₂₇ and R₂₈ are OR₂₉.

For example, at least one of R₂₇ and R₂₈ is OR₂₉.

For example, each R₂₉ independently is H.

For example, each R₂₉ independently is C₁-C₃ alkyl, e.g., methyl.

For example, each R₂₉ independently is C₁-C₃ alkyl substituted with oneor more of C₁-C₆ alkoxyl that is optionally substituted with one or moreOH or OC(O)—C₁-C₆ alkyl.

For example, each R₂₉ independently is CH₂CH₂OCH₃.

For example, each R₂₉ independently is CH(OCH₂CH₂OH)₂.

For example, each R₂₉ independently is CH(OCH₂CH₂OCOCH₃)₂.

For example, each R₂₉ independently is unsubstituted or substitutedC₂-C₆ alkenyl, e.g., propen-3-yl.

For example, each R₂₉ independently is unsubstituted or substitutedC₂-C₆ alkynyl, e.g., propyn-3-yl.

For example, R₂₇ is OCH₂CH₂OCH₃ and R₁ is ethyl substituted withphenoxyl that is substituted with one or more of halo and cyano, e.g.,R₁ being 4-chlorophenoxylethyl, 4-bromophenoxylethyl, or4-cyanophenoxylethyl.

For example, R₂₇ and R₂₈ together form O—R₃₀—O.

For example, R₃₀ is C₁-C₆ alkylene optionally substituted with one ormore of OH, halo, and C₁-C₆ alkoxyl.

For example, R₃₀ is —C(CH₃)₂—, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, or—CH₂CH(CH₃)₂—.

For example, one subset of the compounds of formula (I) includes thoseof formula (Ia) or (Ib):

or a stereoisomer, tautomer or salt thereof.

For example, another subset of the compounds of formula (I) includesthose of formula (IIa) or (IIb):

or a stereoisomer, tautomer or salt thereof.

For example, another subset of the compounds of formula (I) includesthose of formula (IIc), (IId), (IIe), or (IIf):

or a stereoisomer, tautomer or salt thereof.

For example, each of R₂₀, R₂₁, R₂₂, and R₂₃, independently, is -Q₃-T₃.

For example, Q₃ is a bond.

For example, Q₃ is an unsubstituted C₁-C₃ alkyl linker.

For example, T₃ is H or OH.

For example, T₃ is N₃.

For example, T₃ is cyano.

For example, T₃ is NO₂.

For example, T₃ is NH₂.

For example, T₃ is NHCO—C₁-C₆ alkyl, e.g., NHCOCH₃.

For example, T₃ is R_(S3) or OR_(S3) in which R_(S3) is optionallysubstituted C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, or C₆-C₁₀ aryl.

For example, R_(S3) is an unsubstituted or substituted straight chainC₁-C₆ or branched C₃-C₆ alkyl, including but not limited to, methyl,ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyland n-hexyl.

For example, R_(S3) is unsubstituted or substituted C₂-C₆ alkenyl, e.g.,propen-3-yl.

For example, R_(S3) is unsubstituted or substituted C₂-C₆ alkynyl, e.g.,propyn-3-yl.

For example, T₃ is an unsubstituted or substituted straight chain C₁-C₆or branched C₃-C₆ alkyl, including but not limited to, methyl, ethyl,n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl andn-hexyl.

For example, T₃ is optionally substituted C₃-C₆ cycloalkyl, includingbut not limited to, cyclopentyl and cyclohexyl.

For example, T₃ is optionally substituted phenyl.

For example, T₃ is halo (e.g., fluorine, chlorine, bromine, and iodine).

For example, T₃ is optionally substituted 4 to 7-memberedheterocycloalkyl (e.g., azetidinyl, oxetanyl, thietanyl, pyrrolidinyl,imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,triazolidinyl, tetrahyrofuranyl, piperidinyl,1,2,3,6-tetrahydropyridinyl, piperazinyl, tetrahydro-2H-pyranyl,3,6-dihydro-2H-pyranyl, and morpholinyl, and the like).

For example, T₃ is optionally substituted 5 to 6-membered heteroaryl(e.g., pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl,isothiazolyl, and the like).

For example, each of R₂₀, R₂₁, R₂₂, and R₂₃ independently is H, OH,halo, NH₂, cyano, NO₂, N₃, C₁-C₆ alkoxyl, benzyl, or C₁-C₆ alkyloptionally substituted with halo.

For example, each of R₂₀, R₂₁, R₂₂, and R₂₃ independently is H, cyano,N₃, C₁-C₆ alkyl, or benzyl.

For example, one of R₂₀ and R₂₁ is H and the other is R₂₀ is cyano, NO₂,N₃, or C₁-C₃ alkyl.

For example, both R₂₀ and R₂₁ are H.

For example, at least one of R₂₀ and R₂₇ is H.

For example, at least one of R₂₁ and R₂₈ is H.

For example, R₂₂ and R₂₃ are each H.

For example, one of R₂₂ and R₂₃ is H and the other is cyano, NO₂, N₃, orC₁-C₃ alkyl.

For example, at least one of R₂₀, R₂₁, R₂₂, and R₂₃ is not H.

For example, each of R₂₀, R₂₁, R₂₂, and R₂₃ is H.

For example, Y₂ is —CH₂CH₂—.

For example, Y₂ is —CH₂CH₂-Q₀-CH₂CH₂—.

For example, Y₂ is —(CR₄₀R₄₁)_(u-1)—CH(R₄₁)-Q₀-CH(R₄₃)—(CR₄₂R₄₃)_(v-1)—.

For example, u is 1 or 2.

For example, u is 3.

For example, u is 4.

For example, v is 1 or 2.

For example, v is 3.

For example, v is 4.

For example, u is the same as v.

For example, u is different from v.

For example, Q₀ is a bond.

For example, Q₀ is O.

For example, Q₀ is S, SO, or SO₂.

For example, Q₀ is NR₄₄, e.g., NH.

For example, Q₀ is CR₄₅R₄₆.

For example, each of R₄₁ and R₄₃ is H.

For example, each of R₄₀ and R₄₂ is H.

For example, one R₄₁ and one R₄₃, together with the carbon atoms towhich they are attached and Q₀, form C₅-C₈ cycloalkyl, 5- to 8-memberedheterocycloalkyl, phenyl, or 5- to 6-membered heteroaryl, and each ofthe cycloalkyl, heterocycloalkyl, phenyl, or 5- to 6-membered heteroarylis optionally substituted with one or more of OH, halo, cyano, oxo,C₁-C₆ alkyl, or C₁-C₆ haloalkyl.

For example, Y₂ is —CH(R₄₁)-Q₀-CH(R₄₃)—. For example, each of R₄₁ andR₄₃ is H. For example, R₄₁ and R₄₃, together with the carbon atoms towhich they are attached and Q₀, form C₅-C₈ cycloalkyl (e.g.,cyclopentyl, cyclohexyl, and the like). For example, R₄₁ and R₄₃,together with the carbon atoms to which they are attached and Q₀, form5- to 8-membered heterocycloalkyl (e.g., pyrrolidinyl, imidazolidinyl,pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl,tetrahyrofuranyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl, and morpholinyl, and thelike). For example, R₄₁ and R₄₃, together with the carbon atoms to whichthey are attached and Q₀, form phenyl. For example, R₄₁ and R₄₃,together with the carbon atoms to which they are attached and Q₀, form5- to 6-membered heteroaryl (e.g., pyrrolyl, pyrazolyl, imidazolyl,pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, and the like). Forexample, each of said cycloalkyl, heterocycloalkyl, phenyl, or 5- to6-membered heteroaryl is optionally substituted with one or more of OH,halo, cyano, oxo, OP(O)R₄₇R₄₈ (e.g., OP(O)(OH)₂ or OP(O)(F)(OH)), C₁-C₆alkyl, or C₁-C₆ haloalkyl.

For example, Y₂ is —CH₂—CH(R₄₁)-Q₀-CH(R₄₃)—CH₂—. For example, each ofR₄₁ and R₄₃ is H. For example, each of R₄₁ and R₄₃ is OP(O)R₄₇R₄₈, e.g.,OP(O)(OH)₂. For example, R₄₁ and R₄₃, together with the carbon atoms towhich they are attached and Q₀, form C₅-C₈ cycloalkyl (e.g.,cyclopentyl, cyclohexyl, and the like). For example, R₄₁ and R₄₃,together with the carbon atoms to which they are attached and Q₀, form5- to 8-membered heterocycloalkyl (e.g., pyrrolidinyl, imidazolidinyl,pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl,tetrahyrofuranyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl, and morpholinyl, and thelike). For example, R₄₁ and R₄₃, together with the carbon atoms to whichthey are attached and Q₀, form phenyl. For example, R₄₁ and R₄₃,together with the carbon atoms to which they are attached and Q₀, form5- to 6-membered heteroaryl (e.g., pyrrolyl, pyrazolyl, imidazolyl,pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, and the like). Forexample, each of said cycloalkyl, heterocycloalkyl, phenyl, or 5- to6-membered heteroaryl is optionally substituted with one or more of OH,halo, cyano, oxo, OP(O)R₄₇R₄₈ (e.g., OP(O)(OH)₂ or OP(O)(F)(OH)), C₁-C₆alkyl, or C₁-C₆ haloalkyl.

For example, R₄₁ and R₄₃, together with the carbon atoms to which theyare attached and Q₀, form 1,3-cyclohexyl, 2,6-tetrahydropyranyl,2,6-tetrahydropyranyl, or 2,5-thiazolyl, each of which is optionallysubstituted with one or more OH.

For example, R₄₄ is C₁-C₆ alkyl.

For example, R₄₄ is H.

For example, R₄₄ is an amine protecting group (e.g.,t-butyloxylcarbonyl).

For example, each of R₄₅ and R₄₆ is H.

For example, at least one of R₄₅ and R₄₆ is OP(O)R₄₇R₄₈, or C₁-C₆ alkyloptionally substituted with one or more OP(O)R₄₇R₄₈.

For example, at least one of R₄₇ and R₄₈ is halo, e.g., F, Cl, Br or I.

For example, at least one of R₄₇ and R₄₈ is OH.

For example, one of R₄₅ and R₄₆ is H and the other is OP(O)(OH)₂.

For example, one of R₄₅ and R₄₆ is H and the other is OP(O)(F)(OH).

For example, one of R₄₅ and R₄₆ is H and the other is C₁-C₆ alkyloptionally substituted with one or more OP(O)R₄₇R₄₈, e.g., OP(O)(OH)₂.

For example, each of R₄₅ and R₄₆ independently is C₁-C₆ alkyl optionallysubstituted with one or more OP(O)R₄₇R₄₈.

For example, each of R₄₅ and R₄₆ independently is C₁-C₆ alkyl optionallysubstituted with one or more OP(O)(OH)₂, e.g., —CH₂—OP(O)(OH)₂.

For example, each of R₄₅ and R₄₆ independently is C₁-C₆ alkyl optionallysubstituted with one or more OP(O)(F)(OH), e.g., —CH₂—OP(O)(F)(OH).

In embodiments, the variables in formulae (Ia), (Ib) and (IIa)-(IIf) areas defined herein for formula (I), where applicable.

In embodiments, the compounds of any of formulae (I), (Ia), (Ib) and(IIa)-(IIf) are cap analogs. In embodiments, the compounds of any offormulae (I), (Ia), (Ib) and (IIa)-(IIf) are anti-reverse cap analogs(ARCAs). In embodiments, a compound of any of formulae (I), (Ia), (Ib)and (IIa)-(IIf) is incorporated in an RNA molecule (e.g., mRNA) at the5′ end.

In yet another aspect, the present disclosure also provides a compound(e.g., a cap analog) or a polynucleotide containing the cap analoghaving an improved eIF4E binding affinity, enhanced resistance todegradation, or both, as compared to, e.g., natural mRNA caps andnatural mRNAs. As used herein, k_(off) is the off-rate, calculated fromthe dissociation phase, k_(on n) is the on-rate, calculated from theassociation phase; K_(d) or K_(D) is the binding affinity, which is theratio of k_(off)/k_(on), and the residence time, τ, is the inverse ofk_(off).

In embodiments, the compound with an improved eIF4E binding affinity hasa residence time, τ, of about 2 seconds or longer when binding with theeukaryotic initiation factor 4E (eIF4E) characterized by surface plasmonresonance (SPR). For example, τ of the compound is 5 seconds, 10seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 50 seconds, 75seconds, 80 seconds, 90 seconds, 100 seconds, or longer. For example,the compound has an eIF4E k_(off) of no more than 1 s⁻¹ (e.g., no morethan 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.08, 0.06, 0.04,0.02, or 0.01 s⁻¹). For example, the compound having τ of about 2seconds or longer (e.g., 5 seconds, 10 seconds, 15 seconds, 20 seconds,25 seconds, 30 seconds, 50 seconds, 75 seconds, 80 seconds, 90 seconds,100 seconds, or longer) is a compound of any of formulae (I), (Ia), (Ib)and (IIa)-(IIf) or a derivative or analog thereof. For example, thecompound having τ of about 2 seconds or longer (e.g., 5 seconds, 10seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 50 seconds, 75seconds, 80 seconds, 90 seconds, 100 seconds, or longer) is selectedfrom any of those included in Tables 1-2, and stereoisomers, tautomersand salts thereof.

In embodiments, the compound with an improved eIF4E binding affinity hasa residence time, τ, of at least 2 times of that of a natural cap whenbinding with eIF4E characterized by surface plasmon resonance (SPR). Forexample, τ of the compound is at least 3, 4, 5, 6, 7, 10, 15, 20, 25,30, 40, 50, 60, 70, 80, 90, or 100 times of that of a natural cap. Forexample, the compound having τ of at least 2 times (e.g., at least 3, 4,5, 6, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 times) ofthat of a natural cap is a compound of any of formulae (I), (Ia), (Ib)and (IIa)-(IIf) or a derivative or analog thereof. For example, thecompound having τ of at least 2 times (e.g., at least 3, 4, 5, 6, 7, 10,15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 times) of that of anatural cap is selected from any of those included in Tables 1-2, andstereoisomers, tautomers and salts thereof.

In embodiments, the compound with an improved eIF4E binding affinity hasa K_(d) or K_(D) of no more than 10 μM, e.g., using SPR. For example,K_(d) of the compound is no more than 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9,0.7, 0.5, 0.3, or 0.1 μM. For example, the compound has an eIF4E K_(d)of no more than 10 μM (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2, 1,0.9, 0.7, 0.5, 0.3, or 0.1 μM) and a τ of about 2 seconds or longer(e.g., 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30seconds, 50 seconds, 75 seconds, 80 seconds, 90 seconds, 100 seconds, orlonger). For example, the compound having K_(d) of no more than 10 μM(e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.7, 0.5, 0.3, or0.1 μM) is a compound of any of formulae (I), (Ia), (Ib) and (IIa)-(IIf)or a derivative or analog thereof. For example, the compound havingK_(d) of no more than 10 μM (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2,1, 0.9, 0.7, 0.5, 0.3, or 0.1 μM) is selected from any of those includedin Tables 1-2, and stereoisomers, tautomers and salts thereof.

In embodiments, the RNA molecule carrying the compound (e.g., a capanalog) disclosed herein has enhanced resistance to degradation. Forexample, the modified RNA molecule has a half-life that is at least 1.2times of that of a corresponding natural RNA molecule in a cellularenvironment. For example, the half-life of the modified RNA molecule isat least 1.5, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80,90, or 100 times of that of a corresponding natural RNA molecule in acellular environment. For example, the modified RNA molecule carries acompound of any of formulae (I), (Ia), (Ib) and (IIa)-(IIf) or aderivative or analog thereof. For example, the modified RNA moleculecarries a compound selected from any of those included in Tables 1-2,and stereoisomers, tautomers and salts thereof.

Representative compounds of the present disclosure include compoundslisted in Tables 1 and 2 and stereoisomer, tautomer, and salts thereof.

TABLE 1

Cpd No. Y₂ X₁ 1 —CH₂CH₂— N⁺(CH₃) 2 —CH₂CH₂—O—CH₂CH₂— N⁺(CH₃) 3—CH₂CH₂—S—CH₂CH₂— N⁺(CH₃) 4 —CH₂CH₂—S(O)—CH₂CH₂— N⁺(CH₃) 5—CH₂CH₂—S(O)₂—CH₂CH₂— N⁺(CH₃) 6 —CH₂CH₂—NH—CH₂CH₂— N⁺(CH₃) 7

N⁺(CH₃) 8

N⁺(CH₃) 9

N⁺(CH₃) 10

N⁺(CH₃) 11

N⁺(CH₃) 12

N⁺(CH₃) 13 —CH₂CH₂— N 14 —CH₂CH₂—O—CH₂CH₂— N 15 —CH₂CH₂—S—CH₂CH₂— N 16—CH₂CH₂—S(O)—CH₂CH₂— N 17 —CH₂CH₂—S(O)₂—CH₂CH₂— N 18 —CH₂CH₂—NH—CH₂CH₂—N 19

N 20

N 21

N 22

N 23

N 24

N 25

N⁺(CH₃) 26

N⁺(CH₃) 27

N⁺(CH₃) 28

N⁺(CH₃) 29

N⁺(CH₃) 30

N 31

N 32

N 33

N 34

N

TABLE 2 Cpd No. Structure 35

36

37

38

39

40

41

42

43

For example, the compounds listed in Tables 1 and 2 can or may have B₁ring being replaced with any of those as defined in formula (I), e.g.,those with R₁ being 4-chlorophenoxylethyl, 4-bromophenoxylethyl, or4-cyanophenoxylethyl. Alternatively or additionally, the compoundslisted in Tables 1-2 can or may have B₂ ring being replaced with any ofthose as defined in formula (I), e.g., unmodified or modified cytosineor uracil. As another example, the compounds listed in Tables 1-2 can ormay have R₂ (e.g., OH) being replaced with any of those as defined informula (I), e.g., OCH₃, OCH(OCH₂CH₂OH)₂ or OCH(OCH₂CH₂OCOCH₃)₂.

As used herein, the term “LNA” or “locked nucleic acid” refers to amethylene bridge between the 2′O and 4′C of the nucleotide monomer andit also refers to a sugar analog, a nucleoside, a nucleotide monomer, ora nucleic acid, each of which contains such bridge. For example, LNA hasthe following structure

or those described in WO 99/14226 and Kore et al., J. AM CHEM. SOC.2009, 131, 6364-6365, the contents of each of which are incorporatedherein by reference in their entireties.

As used herein, the term “nucleobase” refers to a nitrogen-containingheterocyclic moiety, which is the parts of the nucleic acids that areinvolved in the hydrogen-bonding that binds one nucleic acid strand toanother complementary strand in a sequence specific manner. The mostcommon naturally-occurring nucleobases are: adenine (A), cytosine (C),guanine (G), thymine (T), and uracil (U).

The term “modified nucleobase” refers to a moiety that can replace anucleobase. The modified nucleobase mimics the spatial arrangement,electronic properties, or some other physicochemical property of thenucleobase and retains the property of hydrogen-bonding that binds onenucleic acid strand to another in a sequence specific manner. A modifiednucleobase can pair with at least one of the five naturally occurringbases (uracil, thymine, adenine, cytosine, or guanine) withoutsubstantially affecting the melting behavior, recognition byintracellular enzymes, or activity of the oligonucleotide duplex. Theterm “modified nucleoside” or “modified nucleotide” refers to anucleoside or nucleotide that contains a modified nucleobase and/orother chemical modification disclosed herein, such as modified sugar,modified phosphorus atom bridges or modified internucleoside linkage.

Non-limiting examples of suitable nucleobases include, but are notlimited to, uracil, thymine, adenine, cytosine, and guanine optionallyhaving their respective amino groups protected by, e.g., acyl protectinggroups, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine,2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil,2,6-diaminopurine, azacytosine, 2-thiouracil, 2-thiothymine,2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine),hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine),N8-(8-aza-7-deazaadenine), pyrimidine analogs such as pseudoisocytosineand pseudouracil and other modified nucleobases such as 8-substitutedpurines, xanthine, or hypoxanthine (the latter two being the naturaldegradation products). Exemplary modified nucleobases are disclosed inChiu and Rana, R N A, 2003, 9, 1034-1048, Limbach et al. Nucleic AcidsResearch, 1994, 22, 2183-2196 and Revankar and Rao, ComprehensiveNatural Products Chemistry, vol. 7, 313.

Compounds represented by the following general formulae are alsocontemplated as nucleobases:

in which R₁ and X₁ are as defined herein,

each of R₁₀₀ and R₁₀₁ independently is H, C₁-C₆ alkyl, or an amineprotecting group (such as —C(O)R′ in which R′ is an optionallysubstituted, linear or branched group selected from aliphatic, aryl,aralkyl, aryloxylalkyl, carbocyclyl, heterocyclyl or heteroaryl grouphaving 1 to 15 carbon atoms, including, by way of example only, amethyl, isopropyl, phenyl, benzyl, or phenoxymethyl group), or R₁₀₀ andR₁₀₁ together with the N atom to which they are attached form—N═CH—NR′R″ in which each of R′ and R″ is independently an optionallysubstituted aliphatic, carbocyclyl, aryl, heterocyclyl or heteroaryl; orR₁₀₀ and R₁₀₁ together with the N atom to which they are attached form a4 to 12-membered heterocycloalkyl (e.g., phthalimidyl optionallysubstituted with one or more substituents selected from OH and halo),—N═CH—R₁₀₃, or —N═N—R₁₀₃, wherein R₁₀₃ is phenyl, and each of the 4 to12-membered heterocycloalkyl and R₁₀₃ is optionally substituted with oneor more substituents selected from OH, oxo, halo, C₁-C₆ alkyl, COOH,C(O)O—C₁-C₆ alkyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino,and di-C₁-C₆ alkylamino; and

each R₁₀₂ independently is H, NH₂, or C₁-C₆ alkyl; or R₁₀₂ and one ofR₁₀₀ and R₁₀₁, together with the two nitrogen atoms to which they attachand the carbon atom connecting the two nitrogen atoms form a 5- or6-membered heterocycle which is optionally substituted with one or moreof OH, halo, C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl, or astereoisomer, tautomer or salt thereof. For example, the other of R₁₀₀and R₁₀₁ that does not form the heterocycle is absent, H, or C₁-C₆alkyl.

Modified nucleobases also include expanded-size nucleobases in which oneor more aryl rings, such as phenyl rings, have been added. Some examplesof these expanded-size nucleobases are shown below:

The term “modified sugar” or “sugar analog” refers to a moiety that canreplace a sugar. The modified sugar mimics the spatial arrangement,electronic properties, or some other physicochemical property of asugar.

As used herein, the terms “polynucleotide”, “oligonucleotide” and“nucleic acid” are used interchangeably and refer to single stranded anddouble stranded polymers or oligomers of nucleotide monomers, includingribonucleotides (RNA) and 2′-deoxyribonucleotides (DNA) linked byinternucleotide phosphodiester bond linkages. A polynucleotide may becomposed entirely of deoxyribonucleotides, entirely of ribonucleotidesor chimeric mixtures thereof.

As used herein, the term “messenger RNA” (mRNA) refers to anypolynucleotide which encodes at least one peptide or polypeptide ofinterest and which is capable of being translated to produce the encodedpeptide polypeptide of interest in vitro, in vivo, in situ or ex vivo.An mRNA has been transcribed from a DNA sequence by an RNA polymeraseenzyme, and interacts with a ribosome to synthesize genetic informationencoded by DNA. Generally, mRNA are classified into two sub-classes:pre-mRNA and mature mRNA. Precursor mRNA (pre-mRNA) is mRNA that hasbeen transcribed by RNA polymerase but has not undergone anypost-transcriptional processing (e.g., 5′capping, splicing, editing, andpolyadenylation). Mature mRNA has been modified via post-transcriptionalprocessing (e.g., spliced to remove introns and polyadenylated) and iscapable of interacting with ribosomes to perform protein synthesis. mRNAcan be isolated from tissues or cells by a variety of methods. Forexample, a total RNA extraction can be performed on cells or a celllysate and the resulting extracted total RNA can be purified (e.g., on acolumn comprising oligo-dT beads) to obtain extracted mRNA.

Alternatively, mRNA can be synthesized in a cell-free environment, forexample by in vitro transcription (IVT). An “in vitro transcriptiontemplate” as used herein, refers to deoxyribonucleic acid (DNA) suitablefor use in an IVT reaction for the production of messenger RNA (mRNA).In some embodiments, an IVT template encodes a 5′ untranslated region,contains an open reading frame, and encodes a 3′ untranslated region anda polyA tail. The particular nucleotide sequence composition and lengthof an IVT template will depend on the mRNA of interest encoded by thetemplate.

A “5′ untranslated region (UTR)” refers to a region of an mRNA that isdirectly upstream (i.e., 5′) from the start codon (i.e., the first codonof an mRNA transcript translated by a ribosome) that does not encode aprotein or peptide.

A “3′ untranslated region (UTR)” refers to a region of an mRNA that isdirectly downstream (i.e., 3′) from the stop codon (i.e., the codon ofan mRNA transcript that signals a termination of translation) that doesnot encode a protein or peptide.

An “open reading frame” is a continuous stretch of DNA beginning with astart codon (e.g., methionine (ATG)), and ending with a stop codon(e.g., TAA, TAG or TGA) and encodes a protein or peptide.

A “polyA tail” is a region of mRNA that is downstream, e.g., directlydownstream (i.e., 3′), from the 3′ UTR that contains multiple,consecutive adenosine monophosphates. A polyA tail may contain 10 to 300adenosine monophosphates. For example, a polyA tail may contain 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosinemonophosphates. In some embodiments, a polyA tail contains 50 to 250adenosine monophosphates. In a relevant biological setting (e.g., incells, in vivo, etc.) the poly(A) tail functions to protect mRNA fromenzymatic degradation, e.g., in the cytoplasm, and aids in transcriptiontermination, export of the mRNA from the nucleus, and translation.

Thus, the polynucleotide may in some embodiments comprise (a) a firstregion of linked nucleosides encoding a polypeptide of interest; (b) afirst terminal region located 5′ relative to said first regioncomprising a 5′ untranslated region (UTR); (c) a second terminal regionlocated 3′ relative to said first region; and (d) a tailing region. Theterms polynucleotide and nucleic acid are used interchangeably herein.

In some embodiments, the polynucleotide includes from about 200 to about3,000 nucleotides (e.g., from 200 to 500, from 200 to 1,000, from 200 to1,500, from 200 to 3,000, from 500 to 1,000, from 500 to 1,500, from 500to 2,000, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000,from 1,000 to 3,000, from 1,500 to 3,000, or from 2,000 to 3,000nucleotides).

IVT mRNA disclosed herein may function as mRNA but are distinguishedfrom wild-type mRNA in their functional and/or structural designfeatures which serve to overcome existing problems of effectivepolypeptide production using nucleic-acid based therapeutics. Forexample, IVT mRNA may be structurally modified or chemically modified.As used herein, a “structural” modification is one in which two or morelinked nucleosides are inserted, deleted, duplicated, inverted orrandomized in a polynucleotide without significant chemical modificationto the nucleotides themselves. Because chemical bonds will necessarilybe broken and reformed to effect a structural modification, structuralmodifications are of a chemical nature and hence are chemicalmodifications. However, structural modifications will result in adifferent sequence of nucleotides. For example, the polynucleotide“ATCG” may be chemically modified to “AT-5meC-G”. The samepolynucleotide may be structurally modified from “ATCG” to “ATCCCG”.Here, the dinucleotide “CC” has been inserted, resulting in a structuralmodification to the polynucleotide.

cDNA encoding the polynucleotides described herein may be transcribedusing an in vitro transcription (IVT) system. The system typicallycomprises a transcription buffer, nucleotide triphosphates (NTPs), anRNase inhibitor and a polymerase. The NTPs may be manufactured in house,may be selected from a supplier, or may be synthesized as describedherein. The NTPs may be selected from, but are not limited to, thosedescribed herein including natural and unnatural (modified) NTPs. Thepolymerase may be selected from, but is not limited to, T₇ RNApolymerase, T₃ RNA polymerase and mutant polymerases such as, but notlimited to, polymerases able to incorporate polynucleotides (e.g.,modified nucleic acids). TP as used herein stands for triphosphate.

In embodiments, polynucleotides of the disclosure may include at leastone chemical modification. The polynucleotides described herein caninclude various substitutions and/or insertions from native or naturallyoccurring polynucleotides, e.g., in addition to the modification on the5′ terminal mRNA cap moieties disclosed herein. As used herein, whenreferring to a polynucleotide, the terms “chemical modification” or, asappropriate, “chemically modified” refer to modification with respect toadenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C)ribo- or deoxyribnucleosides and the internucleoside linkages in one ormore of their position, pattern, percent or population. Generally,herein, these terms are not intended to refer to the ribonucleotidemodifications in naturally occurring 5′-terminal mRNA cap moieties.

The modifications may be various distinct modifications. In someembodiments, the regions may contain one, two, or more (optionallydifferent) nucleoside or nucleotide modifications. In some embodiments,a modified polynucleotide introduced to a cell may exhibit reduceddegradation in the cell as compared to an unmodified polynucleotide.

Modifications of the polynucleotides of the disclosure include, but arenot limited to those listed in detail below. The polynucleotide maycomprise modifications which are naturally occurring, non-naturallyoccurring or the polynucleotide can comprise both naturally andnon-naturally occurring modifications.

The polynucleotides of the disclosure can include any modification, suchas to the sugar, the nucleobase, or the internucleoside linkage (e.g.,to a linking phosphate/to a phosphodiester linkage/to the phosphodiesterbackbone). One or more atoms of a pyrimidine or purine nucleobase may bereplaced or substituted with optionally substituted amino, optionallysubstituted thiol, optionally substituted alkyl (e.g., methyl or ethyl),or halo (e.g., chloro or fluoro).

In certain embodiments, modifications (e.g., one or more modifications)are present in each of the sugar and the internucleoside linkage.Modifications according to the present disclosure may be modificationsof ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threosenucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids(PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additionalmodifications are described herein.

Non-natural modified nucleotides may be introduced to polynucleotidesduring synthesis or post-synthesis of the chains to achieve desiredfunctions or properties. The modifications may be on internucleotidelineage, the purine or pyrimidine bases, or sugar. The modification maybe introduced at the terminal of a chain or anywhere else in the chain;with chemical synthesis or with a polymerase enzyme. Any of the regionsof the polynucleotides may be chemically modified.

The present disclosure provides for polynucleotides comprised ofunmodified or modified nucleosides and nucleotides and combinationsthereof. As described herein “nucleoside” is defined as a compoundcontaining a sugar molecule (e.g., a pentose or ribose) or a derivativethereof in combination with an organic base (e.g., a purine orpyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). As described herein, “nucleotide” is defined as anucleoside including a phosphate group. The modified nucleotides may bysynthesized by any useful method, as described herein (e.g., chemically,enzymatically, or recombinantly to include one or more modified ornon-natural nucleosides). The polynucleotides may comprise a region orregions of linked nucleosides. Such regions may have variable backbonelinkages. The linkages may be standard phosphodiester linkages, in whichcase the polynucleotides would comprise regions of nucleotides. Anycombination of base/sugar or linker may be incorporated into thepolynucleotides of the disclosure.

Modifications of polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides), including but not limited to chemicalmodification, that are useful in the compositions, methods and syntheticprocesses of the present disclosure include, but are not limited to thefollowing: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine;2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycinylcarbamoyladenosine;N6-isopentenyladenosine; N6-methyladenosine;N6-threonylcarbamoyladenosine; 1,2′-O-dimethyladenosine;1-methyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine(phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine;2-methylthio-N6-hydroxynorvalyl carbamoyladenosine;2′-O-methyladenosine; 2′-O-ribosyladenosine (phosphate);Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine;N6,2′-O-dimethyladenosine; N6,2′-O-dimethyladenosine;N6,N6,2′-O-trimethyladenosine; N6,N6-dimethyladenosine;N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine;N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine;2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine;N1-methyl-adenosine; N6,N6 (dimethyl)adenine;N6-cis-hydroxy-isopentenyl-adenosine; α-thio-adenosine; 2(amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine;2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine;2-(propyl)adenine; 2′-Amino-2′-deoxy-ATP; 2′-Azido-2′-deoxy-ATP;2′-Deoxy-2′-a-aminoadenosine TP; 2′-Deoxy-2′-a-azidoadenosine TP; 6(alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine;7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8(amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine;8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine;8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine;8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine;N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine;1-Deazaadenosine TP; 2′Fluoro-N6-Bz-deoxyadenosine TP;2′-OMe-2-Amino-ATP; 2′O-methyl-N6-Bz-deoxyadenosine TP;2′-a-Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP;2-Amino-ATP; 2′-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP;2′-b-Ethynyladenosine TP; 2-Bromoadenosine TP;2′-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP;2′-Deoxy-2′,2′-difluoroadenosine TP; 2′-Deoxy-2′-a-mercaptoadenosine TP;2′-Deoxy-2′-a-thiomethoxyadenosine TP; 2′-Deoxy-2′-b-aminoadenosine TP;2′-Deoxy-2′-b-azidoadenosine TP; 2′-Deoxy-2′-b-bromoadenosine TP;2′-Deoxy-2′-b-chloroadenosine TP; 2′-Deoxy-2′-b-fluoroadenosine TP;2′-Deoxy-2′-b-iodoadenosine TP; 2′-Deoxy-2′-b-mercaptoadenosine TP;2′-Deoxy-2′-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP;2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine;2-methylthio-adenine; 2-Trifluoromethyladenosine TP;3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP;3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP;3-Deazaadenosine TP; 4′-Azidoadenosine TP; 4′-Carbocyclic adenosine TP;4′-Ethynyladenosine TP; 5′-Homo-adenosine TP; 8-Aza-ATP;8-bromoadenosine TP; 8-Trifluoromethyladenosine TP; 9-Deazaadenosine TP;2-aminopurine; 7-deaza-2,6-diaminopurine;7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine;2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine;2-thiocytidine; 3-methylcytidine; 5-formylcytidine;5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine;2′-O-methylcytidine; 2′-O-methylcytidine; 5,2′-O-dimethylcytidine;5-formyl-2′-O-methylcytidine; Lysidine; N4,2′-O-dimethylcytidine;N4-acetyl-2′-O-methylcytidine; N4-methylcytidine;N4,N4-Dimethyl-2′-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine;Pseudo-iso-cytidine; pyrrolo-cytidine; α-thio-cytidine;2-(thio)cytosine; 2′-Amino-2′-deoxy-CTP; 2′-Azido-2′-deoxy-CTP;2′-Deoxy-2′-a-aminocytidine TP; 2′-Deoxy-2′-a-azidocytidine TP; 3(deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine;3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2′-O-dimethylcytidine;5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine;5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine;5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine;6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine;1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine;2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine;2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine;4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine;4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine;5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine;Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2′-anhydro-cytidine TPhydrochloride; 2′Fluor-N4-Bz-cytidine TP; 2′Fluoro-N4-Acetyl-cytidineTP; 2′-O-Methyl-N4-Acetyl-cytidine TP; 2′O-methyl-N4-Bz-cytidine TP;2′-a-Ethynylcytidine TP; 2′-a-Trifluoromethylcytidine TP;2′-b-Ethynylcytidine TP; 2′-b-Trifluoromethylcytidine TP;2′-Deoxy-2′,2′-difluorocytidine TP; 2′-Deoxy-2′-a-mercaptocytidine TP;2′-Deoxy-2′-a-thiomethoxycytidine TP; 2′-Deoxy-2′-b-aminocytidine TP;2′-Deoxy-2′-b-azidocytidine TP; 2′-Deoxy-2′-b-bromocytidine TP;2′-Deoxy-2′-b-chlorocytidine TP; 2′-Deoxy-2′-b-fluorocytidine TP;2′-Deoxy-2′-b-iodocytidine TP; 2′-Deoxy-2′-b-mercaptocytidine TP;2′-Deoxy-2′-b-thiomethoxycytidine TP; 2′-O-Methyl-5-(1-propynyl)cytidineTP; 3′-Ethynylcytidine TP; 4′-Azidocytidine TP; 4′-Carbocyclic cytidineTP; 4′-Ethynylcytidine TP; 5-(1-Propynyl)ara-cytidine TP;5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidineTP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP;5-Ethynylcytidine TP; 5′-Homo-cytidine TP; 5-Methoxycytidine TP;5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidineTP; Pseudoisocytidine; 7-methylguanosine; N2,2′-O-dimethylguanosine;N2-methylguanosine; Wyosine; 1,2′-O-dimethylguanosine;1-methylguanosine; 2′-O-methylguanosine; 2′-O-ribosylguanosine(phosphate); 2′-O-methylguanosine; 2′-O-ribosylguanosine (phosphate);7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine;Methylwyosine; N2,7-dimethylguanosine; N2,N2,2′-O-trimethylguanosine;N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine;N2,7,2′-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine;8-oxo-guanosine; N1-methyl-guanosine; α-thio-guanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2′-Amino-2′-deoxy-GTP;2′-Azido-2′-deoxy-GTP; 2′-Deoxy-2′-a-aminoguanosine TP;2′-Deoxy-2′-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine;6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7(deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine;7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8(halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine;8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine;8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; azaguanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine;1-methyl-6-thio-guanosine; 6-methoxy-guanosine;6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine;6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine;7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine;N2-methyl-6-thio-guanosine; 1-Me-GTP; 2′Fluoro-N2-isobutyl-guanosine TP;2′O-methyl-N2-isobutyl-guanosine TP; 2′-a-Ethynylguanosine TP;2′-a-Trifluoromethylguanosine TP; 2′-b-Ethynylguanosine TP;2′-b-Trifluoromethylguanosine TP; 2′-Deoxy-2′,2′-difluoroguanosine TP;2′-Deoxy-2′-a-mercaptoguanosine TP; 2′-Deoxy-2′-a-thiomethoxyguanosineTP; 2′-Deoxy-2′-b-aminoguanosine TP; 2′-Deoxy-2′-b-azidoguanosine TP;2′-Deoxy-2′-b-bromoguanosine TP; 2′-Deoxy-2′-b-chloroguanosine TP;2′-Deoxy-2′-b-fluoroguanosine TP; 2′-Deoxy-2′-b-iodoguanosine TP;2′-Deoxy-2′-b-mercaptoguanosine TP; 2′-Deoxy-2′-b-thiomethoxyguanosineTP; 4′-Azidoguanosine TP; 4′-Carbocyclic guanosine TP;4′-Ethynylguanosine TP; 5′-Homo-guanosine TP; 8-bromo-guanosine TP;9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine;1,2′-O-dimethylinosine; 2′-O-methylinosine; 7-methylinosine;2′-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine;Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deazathymidine; deoxy-thymidine; 2′-O-methyluridine; 2-thiouridine;3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine;5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine;Dihydrouridine; Pseudouridine; (3-(3-amino-3-carboxypropyl)uridine;1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine;1-methylpseduouridine; 1-ethyl-pseudouridine; 2′-O-methyluridine;2′-O-methylpseudouridine; 2′—O-methyluridine; 2-thio-2′-O-methyluridine;3-(3-amino-3-carboxypropyl)uridine; 3,2′-O-dimethyluridine;3-Methyl-pseudo-Uridine TP; 4-thiouridine;5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methylester; 5,2′-O-dimethyluridine; 5,6-dihydro-uridine;5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2′-O-methyluridine;5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine;5-carboxyhydroxymethyluridine methyl ester;5-carboxymethylaminomethyl-2′-O-methyluridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine;5-Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2′-O-methyluridine;5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine;5-methyluridine), 5-methoxyuridine; 5-methyl-2-thiouridine;5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine;5-methylaminomethyluridine; 5-Methyldihydrouridine; 5-Oxyaceticacid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP;N1-methyl-pseudo-uracil; N1-ethyl-pseudo-uracil; uridine 5-oxyaceticacid; uridine 5-oxyacetic acid methyl ester;3-(3-Amino-3-carboxypropyl)-Uridine TP;5-(iso-Pentenylaminomethyl)-2-thiouridine TP;5-(iso-Pentenylaminomethyl)-2′-O-methyluridine TP;5-(iso-PentenylaminomethyOuridine TP; 5-propynyl uracil; α-thio-uridine;1 (aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1(aminocarbonylethylenyl)-2(thio)-pseudouracil; 1(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminocarbonylethylenyl)-pseudouracil; 1 substituted2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1substituted 4 (thio)pseudouracil; 1 substituted pseudouracil;1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil;1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP;1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP;1-Ethyl-pseudo-UTP; 2 (thio)pseudouracil; 2′ deoxy uridine; 2′fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2′ methyl,2′amino, 2′azido, 2′fluro-guanosine; 2′-Amino-2′-deoxy-UTP;2′-Azido-2′-deoxy-UTP; 2′-Azido-deoxyuridine TP;2′-O-methylpseudouridine; 2′ deoxy uridine; 2′ fluorouridine;2′-Deoxy-2′-a-aminouridine TP; 2′-Deoxy-2′-a-azidouridine TP;2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4(thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4-thiouracil;5 (1,3-diazole-1-alkyl)uracil; 5 (2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5(guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5(methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl)2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2(thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5(methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil;5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil;5-(alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil;5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil;5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil;5-(guanidiniumalkyl)uracil; 5-(halo)uracil;5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil;5-(methoxycarbonylmethyl)-2-(thio)uracil;5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl)2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil;5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil;5-(methyl)-4 (thio)pseudouracil; 5-(methyl)pseudouracil;5-(methylaminomethyl)-2 (thio)uracil; 5-(methylaminomethyl)-2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil;5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine;5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine;allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil;Pseudo-UTP-1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP;1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine;1-propynyl-uridine; 1-taurinomethyl-1-methyl-uridine;1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine;2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine;2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine;2-thio-dihydropseudouridine; 2-thio-dihydrouridine;2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine;4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine;4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine;(±)1-(2-Hydroxypropyl)pseudouridine TP;(2R)-1-(2-Hydroxypropyl)pseudouridine TP;(2S)-1-(2-Hydroxypropyl)pseudouridine TP;(E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP;(Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP;1-(2,2,2-Trifluoroethyl)-pseudo-UTP;1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP;1-(2,2-Diethoxyethyl)pseudouridine TP;1-(2,4,6-Trimethylbenzyl)pseudouridine TP;1-(2,4,6-Trimethyl-benzyl)pseudo-UTP;1-(2,4,6-Trimethyl-phenyl)pseudo-UTP;1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP;1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine TP;1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP;1-(3,4-Dimethoxybenzyl)pseudouridine TP;1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Amino-propyl)pseudo-UTP;1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP;1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP;1-(4-Amino-buty 1)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP;1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine TP;1-(4-Chlorobenzyl)pseudouridine TP; 1-(4-Fluorobenzyl)pseudouridine TP;1-(4-Iodobenzyl)pseudouridine TP;1-(4-Methanesulfonylbenzyl)pseudouridine TP;1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP;1-(4-Methoxy-phenyl)pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP;1-(4-Methyl-benzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP;1-(4-Nitro-benzyl)pseudo-UTP; 1(4-Nitro-phenyl)pseudo-UTP;1-(4-Thiomethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethylbenzyl)pseudouridine TP;1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP;1,6-Dimethyl-pseudo-UTP;1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridineTP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}pseudouridine TP;1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP;1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP;1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP;1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP;1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP;1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP;1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP;1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP;1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP;1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP;1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP;1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP;1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP;1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP;1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallylpseudouridine TP;1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP;1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP;1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP;1-Methoxymethylpseudouridine TP;1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP;1-Methyl-6-(4-morpholino)-pseudo-UTP;1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substitutedphenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP;1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP;1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP;1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP;1-Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-ethylcarboxylate-pseudo-UTP;1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP;1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP;1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP;1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP;1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP;1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP;1-Methyl-6-trifluoromethoxy-pseudo-UTP;1-Methyl-6-trifluoromethyl-pseudo-UTP; 1-MorpholinomethylpseudouridineTP; 1-Pentyl-pseudo-UTP; 1-Phenyl-pseudo-UTP; 1-PivaloylpseudouridineTP; 1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP;1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP;1-Thiomethoxymethylpseudouridine TP; 1-ThiomorpholinomethylpseudouridineTP; 1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP;1-Vinylpseudouridine TP; 2,2′-anhydro-uridine TP; 2′-bromo-deoxyuridineTP; 2′-F-5-Methyl-2′-deoxy-UTP; 2′-OMe-S-Me-UTP; 2′-OMe-pseudo-UTP;2′-a-Ethynyluridine TP; 2′-a-Trifluoromethyluridine TP;2′-b-Ethynyluridine TP; 2′-b-Trifluoromethyluridine TP;2′-Deoxy-2′,2′-difluorouridine TP; 2′-Deoxy-2′-a-mercaptouridine TP;2′-Deoxy-2′-a-thiomethoxyuridine TP; 2′-Deoxy-2′-b-aminouridine TP;2′-Deoxy-2′-b-azidouridine TP; 2′-Deoxy-2′-b-bromouridine TP;2′-Deoxy-2′-b-chlorouridine TP; 2′-Deoxy-2′-b-fluorouridine TP;2′-Deoxy-2′-b-iodouridine TP; 2′-Deoxy-2′-b-mercaptouridine TP;2′-Deoxy-2′-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine;2-methoxyuridine; 2′-O-Methyl-5-(1-propynyl)uridine TP;3-Alkyl-pseudo-UTP; 4′-Azidouridine TP; 4′-Carbocyclic uridine TP;4′-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridineTP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5′-Homo-uridine TP;5-iodo-2′-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP;5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine TP;5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP;6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP;6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP;6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP;6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP;6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP;6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP;6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP;6-Methoxy-pseudo-UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP;6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP;6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP;6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine1-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoicacid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; PseudouridineTP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxyl}]propionic acid;Pseudouridine TP1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxyl}]propionicacid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionicacid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP1-methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3-propionic acid;Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid;Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid;Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid;Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine;undermodified hydroxywybutosine; 4-demethylwyosine; 2,6-(diamino)purine;1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine;2,4,5-(trimethyl)phenyl; 2′ methyl, 2′amino, 2′azido, 2′fluro-cytidine;2′ methyl, 2′amino, 2′azido, 2′fluro-adenine; 2′methyl, 2′amino,2′azido, 2′fluro-uridine; 2′-amino-2′-deoxyribose;2-amino-6-Chloro-purine; 2-aza-inosinyl; 2′-azido-2′-deoxyribose;2′fluoro-2′-deoxyribose; 2′-fluoro-modified bases; 2′-O-methyl-ribose;2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl;2-pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl;3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole;4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl;5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine;6-(methyl)-7-(aza)indolyl; 6-chloro-purine;6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aza)indolyl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl,propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl;Aminoindolyl; Anthracenyl;bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl;Isocarbostyrilyl; Isoguanisine; N2-substituted purines;N6-methyl-2-amino-purine; N6-substituted purines; N-alkylatedderivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl;Nitroindazolyl; Nitropyrazolyl; Nubularine; O6-substituted purines;O-alkylated derivative;ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP;para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl;Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl;pyridopyrimidin-3-yl; pyridopyrimidin-3-yl,2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl;Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5′-TP;2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine;pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP;Formycin B TP; Pyrrolosine TP; 2′-OH-ara-adenosine TP;2′-OH-ara-cytidine TP; 2′-OH-ara-uridine TP; 2′-OH-ara-guanosine TP;5-(2-carbomethoxyvinyl)uridine TP; andN6-(19-Amino-pentaoxanonadecyl)adenosine TP.

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) include a combination of at least two (e.g., 2, 3,4 or more) of the aforementioned modified nucleobases.

In some embodiments, modified nucleobases in polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) are selected from thegroup consisting of pseudouridine (ψ), 2-thiouridine (s2U),4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methyluridine, 5-methoxyuridine, 2′-O-methyl uridine,1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ),5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), α-thio-guanosine,α-thio-adenosine, 5-cyano uridine, 4′-thio uridine 7-deaza-adenine,1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine(m6A), and 2,6-Diaminopurine, (I), 1-methyl-inosine (m1I), wyosine(imG), methylwyosine (mimG), 7-deaza-guanosine,7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine(preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G),8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8-dimethyladenosine,2-geranylthiouridine, 2-lysidine, 2-selenouridine,3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine,3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine,5-(carboxyhydroxymethyl)-2′-O-methyluridine methyl ester,5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine,5-aminomethyluridine, 5-carbamoylhydroxymethyluridine,5-carbamoylmethyl-2-thiouridine, 5-carboxymethyl-2-thiouridine,5-carboxymethylaminomethyl-2-geranylthiouridine,5-carboxymethylaminomethyl-2-selenouridine, 5-cyanomethyluridine,5-hydroxycytidine, 5-methylaminomethyl-2-geranylthiouridine,7-aminocarboxypropyl-demethylwyosine, 7-aminocarboxypropylwyosine,7-aminocarboxypropylwyosine methyl ester, 8-methyladenosine,N4,N4-dimethylcytidine, N6-formyladenosine, N6-hydroxymethyladenosine,agmatidine, cyclic N6-threonylcarbamoyladenosine, glutamyl-queuosine,methylated undermodified hydroxywybutosine,N4,N4,2′-O-trimethylcytidine, geranylated5-methylaminomethyl-2-thiouridine, geranylated5-carboxymethylaminomethyl-2-thiouridine, Qbase, preQ0base, preQ1base,and two or more combinations thereof. In some embodiments, the at leastone chemically modified nucleoside is selected from the group consistingof pseudouridine, 1-methyl-pseudouridine, 1-ethyl-pseudouridine,5-methylcytosine, 5-methoxyuridine, and a combination thereof. In someembodiments, the polyribonucleotide (e.g., RNA polyribonucleotide, suchas mRNA polyribonucleotide) includes a combination of at least two(e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases. Insome embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) include a combination of at least two (e.g., 2, 3,4 or more) of the aforementioned modified nucleobases.

In some embodiments, modified nucleobases in polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) are selected from thegroup consisting of 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine(e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine(ψ), α-thio-guanosine and α-thio-adenosine. In some embodiments, thepolyribonucleotide includes a combination of at least two (e.g., 2, 3, 4or more) of the aforementioned modified nucleobases, including but notlimited to chemical modifications.

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) comprise pseudouridine (ψ) and 5-methyl-cytidine(m5C). In some embodiments, the polyribonucleotides (e.g., RNA, such asmRNA) comprise 1-methyl-pseudouridine (m1ψ). In some embodiments, thepolyribonucleotides (e.g., RNA, such as mRNA) comprise1-ethyl-pseudouridine (e1ψ). In some embodiments, thepolyribonucleotides (e.g., RNA, such as mRNA) comprise1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In someembodiments, the polyribonucleotides (e.g., RNA, such as mRNA) comprise1-ethyl-pseudouridine (e1ψ) and 5-methyl-cytidine (m5C). In someembodiments, the polyribonucleotides (e.g., RNA, such as mRNA) comprise2-thiouridine (s2U). In some embodiments, the polyribonucleotides (e.g.,RNA, such as mRNA) comprise 2-thiouridine and 5-methyl-cytidine (m5C).In some embodiments, the polyribonucleotides (e.g., RNA, such as mRNA)comprise methoxy-uridine (mo5U). In some embodiments, thepolyribonucleotides (e.g., RNA, such as mRNA) comprise 5-methoxy-uridine(mo5U) and 5-methyl-cytidine (m5C). In some embodiments, thepolyribonucleotides (e.g., RNA, such as mRNA) comprise 2′-O-methyluridine. In some embodiments, the polyribonucleotides (e.g., RNA, suchas mRNA) comprise 2′-O-methyl uridine and 5-methyl-cytidine (m5C). Insome embodiments, the polyribonucleotides (e.g., RNA, such as mRNA)comprise N6-methyl-adenosine (m6A). In some embodiments, thepolyribonucleotides (e.g., RNA, such as mRNA) compriseN6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) are uniformly modified (e.g., fully modified,modified throughout the entire sequence) for a particular modification.For example, a polynucleotide can be uniformly modified with1-methyl-pseudouridine, meaning that all uridine residues in the mRNAsequence are replaced with 1-methyl-pseudouridine. Similarly, apolynucleotide can be uniformly modified for any type of nucleosideresidue present in the sequence by replacement with a modified residuesuch as those set forth above.

Exemplary nucleobases and nucleosides having a modified cytosine includeN4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C),1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and2-thio-5-methyl-cytidine.

In some embodiments, a modified nucleobase is a modified uridine.Exemplary nucleobases and nucleosides having a modified uridine include1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxyuridine, 2-thio uridine, 5-cyano uridine, 2′-O-methyl uridine and4′-thio uridine.

In some embodiments, a modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), andN6-methyl-adenosine (m6A).

In some embodiments, a modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine(mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0),7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G),1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

The polynucleotides of the present disclosure may be partially or fullymodified along the entire length of the molecule. For example, one ormore or all or a given type of nucleotide (e.g., purine or pyrimidine,or any one or more or all of A, G, U, C) may be uniformly modified in apolynucleotide of the invention, or in a given predetermined sequenceregion thereof (e.g., in the mRNA including or excluding the polyAtail). In some embodiments, all nucleotides X in a polynucleotide of thepresent disclosure (or in a given sequence region thereof) are modifiednucleotides, wherein X may any one of nucleotides A, G, U, C, or any oneof the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C orA+G+C.

The polynucleotide may contain from about 1% to about 100% modifiednucleotides (either in relation to overall nucleotide content, or inrelation to one or more types of nucleotide, i.e., any one or more of A,G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1%to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100%). It will be understood that anyremaining percentage is accounted for by the presence of unmodified A,G, U, or C.

The polynucleotides may contain at a minimum 1% and at maximum 100%modified nucleotides, or any intervening percentage, such as at least 5%modified nucleotides, at least 10% modified nucleotides, at least 25%modified nucleotides, at least 50% modified nucleotides, at least 80%modified nucleotides, or at least 90% modified nucleotides. For example,the polynucleotides may contain a modified pyrimidine such as a modifieduracil or cytosine. In some embodiments, at least 5%, at least 10%, atleast 25%, at least 50%, at least 80%, at least 90% or 100% of theuracil in the polynucleotide is replaced with a modified uracil (e.g., a5-substituted uracil). The modified uracil can be replaced by a compoundhaving a single unique structure, or can be replaced by a plurality ofcompounds having different structures (e.g., 2, 3, 4 or more uniquestructures). In some embodiments, at least 5%, at least 10%, at least25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine inthe polynucleotide is replaced with a modified cytosine (e.g., a5-substituted cytosine). The modified cytosine can be replaced by acompound having a single unique structure, or can be replaced by aplurality of compounds having different structures (e.g., 2, 3, 4 ormore unique structures).

Thus, in some embodiments, the RNA molecules of the invention comprise a5′UTR element, an optionally codon optimized open reading frame, and a3′UTR element, a poly(A) sequence and/or a polyadenylation signalwherein the RNA is not chemically modified.

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyluridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-selenouridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyluridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine,5-taurinomethyl-2-thio-uridine(τm⁵s²U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e.,having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ),1-ethyl-pseudouridine (e1ψ), 5-methyl-2-thio-uridine (m⁵s²U),1-methyl-4-thio-pseudouridine (m¹s⁴ψ), 4-thio-1-methyl-pseudouridine,3-methyl-pseudouridine (m³ψ), 2-thio-1-methyl-pseudouridine,1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine,dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine,5-methyldihydrouridine (m⁵D), 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formyl-cytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k2C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴2 Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms² m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosineonyx)₂-methylthio-N6-threonylcarbamoyl-adenosine (ms² g⁶A),N6,N6-dimethyl-adenosine (m⁶2A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶2Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine (phosphate)(Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine,adenosine, 2′-F-adenosine, 2′-OH-ara-adenosine, andN6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o₂yW), hydroxywybutosine (OhyW), undermodifiedhydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m²2 G), N2,7-dimethyl-guanosine (m^(2,7)G),N2,N2,7-dimethyl-guanosine (m^(2,2,7)G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m²2 Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m²′⁷Gm), 2′-O-methyl-inosine (Im),1,2′-O-dimethyl-inosine (m¹Im), 2′-O-ribosylguanosine (phosphate)(Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2′-F-ara-guanosine, and2′-F-guanosine.

In one embodiment, the polynucleotides of the present disclosure, suchas IVT polynucleotides, may have a uniform chemical modification of allor any of the same nucleoside type or a population of modificationsproduced by mere downward titration of the same starting modification inall or any of the same nucleoside type, or a measured percent of achemical modification of any of the same nucleoside type but with randomincorporation, such as where all uridines are replaced by a uridineanalog, e.g., pseudouridine. In another embodiment, the polynucleotidesmay have a uniform chemical modification of two, three, or four of thenucleoside types throughout the entire polynucleotide (such as both alluridines and all cytosines, etc. are modified in the same way). When thepolynucleotides of the present disclosure are chemically and/orstructurally modified, the polynucleotides may be referred to as“modified polynucleotides.”

As used herein, the term “approximately” or “about,” as applied to oneor more values of interest, refers to a value that is similar to astated reference value, as well as a collection or range of values thatare included. In certain embodiments, the term “approximately” or“about” refers to a range of values that fall within 25%, 20%, 19%, 18%,17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, or less in either direction (greater than or less than) of thestated reference value unless otherwise stated or otherwise evident fromthe context (except where such number would exceed 100% of a possiblevalue). For example, “about X” includes a range of values that are ±20%,±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X, where X is a numericalvalue. In one embodiment, the term “about” refers to a range of valueswhich are 5% more or less than the specified value. In anotherembodiment, the term “about” refers to a range of values which are 2%more or less than the specified value. In another embodiment, the term“about” refers to a range of values which are 1% more or less than thespecified value.

As used herein, “alkyl”, “C₁, C₂, C₃, C₄, C₅ or C₆ alkyl” or “C₁-C₆alkyl” is intended to include C₁, C₂, C₃, C₄, C₅ or C₆ straight chain(linear) saturated aliphatic hydrocarbon groups and C₃, C₄, C₅ or C₆branched saturated aliphatic hydrocarbon groups. For example, C₁-C₆alkyl is intended to include C₁, C₂, C₃, C₄, C₅ and C₆ alkyl groups.Examples of alkyl include, moieties having from one to six carbon atoms,such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, t-butyl, n-pentyl, s-pentyl or n-hexyl.

In certain embodiments, a straight chain or branched alkyl has six orfewer carbon atoms (e.g., C₁-C₆ for straight chain, C₃-C₆ for branchedchain), and in another embodiment, a straight chain or branched alkylhas four or fewer carbon atoms.

As used herein, the term “cycloalkyl” refers to a saturated orunsaturated nonaromatic hydrocarbon mono- or multi-ring (e.g., fused,bridged, or spiro rings) system having 3 to 30 carbon atoms (e.g.,C₃-C₁₀). Examples of cycloalkyl include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and adamantyl.The term “heterocycloalkyl” refers to a saturated or unsaturatednonaromatic 3-8 membered monocyclic, 7-12 membered bicyclic (fused,bridged, or spiro rings), or 11-14 membered tricyclic ring system(fused, bridged, or spiro rings) having one or more heteroatoms (such asO, N, S, or Se), unless specified otherwise. Examples ofheterocycloalkyl groups include, but are not limited to, piperidinyl,piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolinyl,indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,triazolidinyl, oxiranyl, azetidinyl, oxetanyl, thietanyl,1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl, dihydropyranyl, pyranyl,morpholinyl, tetrahydrothiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl,2-oxa-6-azaspiro[3.3]heptanyl, 2,6-diazaspiro[3.3]heptanyl,1,4-dioxa-8-azaspiro[4.5]decanyl, 1,4-dioxaspiro[4.5]decanyl,1-oxaspiro[4.5]decanyl, 1-azaspiro[4.5]decanyl,3′H-spiro[cyclohexane-1,1′-isobenzofuran]-yl,7′H-spiro[cyclohexane-1,5′-furo[3,4-b]pyridin]-yl,3′H-spiro[cyclohexane-1,1′-furo[3,4-c]pyridin]-yl, and the like.

The term “optionally substituted alkyl” refers to unsubstituted alkyl oralkyl having designated substituents replacing one or more hydrogenatoms on one or more carbons of the hydrocarbon backbone. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, acylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

An “arylalkyl” or an “aralkyl” moiety is an alkyl substituted with anaryl (e.g., phenylmethyl (benzyl)). An “alkylaryl” moiety is an arylsubstituted with an alkyl (e.g., methylphenyl).

As used herein, “alkyl linker” is intended to include C₁, C₂, C₃, C₄, C₅or C₆ straight chain (linear) saturated divalent aliphatic hydrocarbongroups and C₃, C₄, C₅ or C₆ branched saturated aliphatic hydrocarbongroups. For example, C₁-C₆ alkyl linker is intended to include C₁, C₂,C₃, C₄, C₅ or C₆ alkyl linker groups. Examples of alkyl linker include,moieties having from one to six carbon atoms, such as, but not limitedto, methyl (—CH₂—), ethyl (—CH₂CH₂—), n-propyl (—CH₂CH₂CH₂—), i-propyl(—CHCH₃CH₂—), n-butyl (—CH₂CH₂CH₂CH₂—), s-butyl (—CHCH₃CH₂CH₂—), i-butyl(—C(CH₃) 2CH₂—), n-pentyl (—CH₂CH₂CH₂CH₂CH₂—), s-pentyl(—CHCH₃CH₂CH₂CH₂—) or n-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₂—).

“Alkenyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double bond. For example, the term “alkenyl” includes straightchain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl,hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branched alkenylgroups.

In certain embodiments, a straight chain or branched alkenyl group hassix or fewer carbon atoms in its backbone (e.g., C₂-C₆ for straightchain, C₃-C₆ for branched chain). The term “C₂-C₆” includes alkenylgroups containing two to six carbon atoms. The term “C₃-C₆” includesalkenyl groups containing three to six carbon atoms.

The term “optionally substituted alkenyl” refers to unsubstitutedalkenyl or alkenyl having designated substituents replacing one or morehydrogen atoms on one or more hydrocarbon backbone carbon atoms. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, acylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

“Alkynyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but which containat least one triple bond. For example, “alkynyl” includes straight chainalkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl,heptynyl, octynyl, nonynyl, decynyl), and branched alkynyl groups. Incertain embodiments, a straight chain or branched alkynyl group has sixor fewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). The term “C₂-C₆” includes alkynyl groupscontaining two to six carbon atoms. The term “C₃-C₆” includes alkynylgroups containing three to six carbon atoms.

The term “optionally substituted alkynyl” refers to unsubstitutedalkynyl or alkynyl having designated substituents replacing one or morehydrogen atoms on one or more hydrocarbon backbone carbon atoms. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, acylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Other optionally substituted moieties (such as optionally substitutedcycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both theunsubstituted moieties and the moieties having one or more of thedesignated substituents. For example, substituted heterocycloalkylincludes those substituted with one or more alkyl groups, such as2,2,6,6-tetramethyl-piperidinyl and2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.

“Aryl” includes groups with aromaticity, including “conjugated,” ormulticyclic systems with at least one aromatic ring and do not containany heteroatom in the ring structure. Examples include phenyl, benzyl,1,2,3,4-tetrahydronaphthalenyl, etc.

“Heteroaryl” groups are aryl groups, as defined above, except havingfrom one to four heteroatoms in the ring structure, and may also bereferred to as “aryl heterocycles” or “heteroaromatics.” As used herein,the term “heteroaryl” is intended to include a stable 5-, 6-, or7-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclicaromatic heterocyclic ring which consists of carbon atoms and one ormore heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6heteroatoms, or e.g. 1, 2, 3, 4, 5, or 6 heteroatoms, independentlyselected from the group consisting of nitrogen, oxygen and sulfur. Thenitrogen atom may be substituted or unsubstituted (i.e., N or NR whereinR is H or other substituents, as defined). The nitrogen and sulfurheteroatoms may optionally be oxidized (i.e., N→O and S(O)_(p), wherep=1 or 2). It is to be noted that total number of S and O atoms in thearomatic heterocycle is not more than 1.

Examples of heteroaryl groups include pyrrole, furan, thiophene,thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole,oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and thelike.

Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryland heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene,benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, quinoline, isoquinoline, naphthrydine, indole,benzofuran, purine, benzofuran, deazapurine, indolizine.

In the case of multicyclic aromatic rings, only one of the rings needsto be aromatic (e.g., 2,3-dihydroindole), although all of the rings maybe aromatic (e.g., quinoline). The second ring can also be fused orbridged.

The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring can besubstituted at one or more ring positions (e.g., the ring-forming carbonor heteroatom such as N) with such substituents as described above, forexample, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl,aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety. Aryl and heteroarylgroups can also be fused or bridged with alicyclic or heterocyclicrings, which are not aromatic so as to form a multicyclic system (e.g.,tetralin, methylenedioxyphenyl such as benzo[d][1,3]dioxole-5-yl).

As used herein, “carbocycle” or “carbocyclic ring” is intended toinclude any stable monocyclic, bicyclic or tricyclic ring having thespecified number of carbons, any of which may be saturated, unsaturated,or aromatic. Carbocycle includes cycloalkyl and aryl. For example, aC₃-C₁₄ carbocycle is intended to include a monocyclic, bicyclic ortricyclic ring having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbonatoms. Examples of carbocycles include, but are not limited to,cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl,cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl, adamantyl,cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl, naphthyl,indanyl, adamantyl and tetrahydronaphthyl. Bridged rings are alsoincluded in the definition of carbocycle, including, for example,[3.3.0]bicyclooctane, [4.3.0]bicyclononane, and [4.4.0]bicyclodecane and[2.2.2]bicyclooctane. A bridged ring occurs when one or more carbonatoms link two non-adjacent carbon atoms. In one embodiment, bridgerings are one or two carbon atoms. It is noted that a bridge alwaysconverts a monocyclic ring into a tricyclic ring. When a ring isbridged, the substituents recited for the ring may also be present onthe bridge. Fused (e.g., naphthyl, tetrahydronaphthyl) and spiro ringsare also included.

As used herein, “heterocycle” or “heterocyclic group” includes any ringstructure (saturated, unsaturated, or aromatic) which contains at leastone ring heteroatom (e.g., N, O or S). Heterocycle includesheterocycloalkyl and heteroaryl. Examples of heterocycles include, butare not limited to, morpholine, pyrrolidine, tetrahydrothiophene,piperidine, piperazine, oxetane, pyran, tetrahydropyran, azetidine, andtetrahydrofuran.

Examples of heterocyclic groups include, but are not limited to,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl (e.g.,benzo[d][1,3]dioxole-5-yl), morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl,pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl,pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl.

The term “substituted,” as used herein, means that any one or morehydrogen atoms on the designated atom is replaced with a selection fromthe indicated groups, provided that the designated atom's normal valencyis not exceeded, and that the substitution results in a stable compound.When a substituent is oxo or keto (i.e., ═O), then 2 hydrogen atoms onthe atom are replaced. Keto substituents are not present on aromaticmoieties. Ring double bonds, as used herein, are double bonds that areformed between two adjacent ring atoms (e.g., C═C, C═N or N═N). “Stablecompound” and “stable structure” are meant to indicate a compound thatis sufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchformula. Combinations of substituents and/or variables are permissible,but only if such combinations result in stable compounds.

When any variable (e.g., R₂₉) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to contain 0-2 R₂₉ moieties, then the groupmay contain up to two R₂₉ moieties and R₂₉ at each occurrence isselected independently from the definition of R₂₉. Also, combinations ofsubstituents and/or variables are permissible, but only if suchcombinations result in stable compounds.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O—.

As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo andiodo. The term “perhalogenated” generally refers to a moiety wherein allhydrogen atoms are replaced by halogen atoms. The term “haloalkyl” or“haloalkoxyl” refers to an alkyl or alkoxyl substituted with one or morehalogen atoms.

The term “carbonyl” includes compounds and moieties which contain acarbon connected with a double bond to an oxygen atom. Examples ofmoieties containing a carbonyl include, but are not limited to,aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “carboxyl” refers to —COOH or its C₁-C₆ alkyl ester.

“Acyl” includes moieties that contain the acyl radical (R—C(O)—) or acarbonyl group. “Substituted acyl” includes acyl groups where one ormore of the hydrogen atoms are replaced by, for example, alkyl groups,alkynyl groups, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

“Aroyl” includes moieties with an aryl or heteroaromatic moiety bound toa carbonyl group. Examples of aroyl groups include phenylcarboxy,naphthyl carboxy, etc.

“Alkoxyalkyl,” “alkylaminoalkyl,” and “thioalkoxyalkyl” include alkylgroups, as described above, wherein oxygen, nitrogen, or sulfur atomsreplace one or more hydrocarbon backbone carbon atoms.

The term “alkoxy” or “alkoxyl” includes substituted and unsubstitutedalkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom.Examples of alkoxy groups or alkoxyl radicals include, but are notlimited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxygroups. Examples of substituted alkoxy groups include halogenated alkoxygroups. The alkoxy groups can be substituted with groups such asalkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.

The term “ether” or “alkoxy” includes compounds or moieties whichcontain an oxygen bonded to two carbon atoms or heteroatoms. Forexample, the term includes “alkoxyalkyl,” which refers to an alkyl,alkenyl, or alkynyl group covalently bonded to an oxygen atom which iscovalently bonded to an alkyl group.

The term “ester” includes compounds or moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc.

The term “thioalkyl” includes compounds or moieties which contain analkyl group connected with a sulfur atom. The thioalkyl groups can besubstituted with groups such as alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, carboxyacid, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “thioether” includes moieties which contain a sulfur atombonded to two carbon atoms or heteroatoms. Examples of thioethersinclude, but are not limited to alkthioalkyls, alkthioalkenyls, andalkthioalkynyls. The term “alkthioalkyls” include moieties with analkyl, alkenyl, or alkynyl group bonded to a sulfur atom which is bondedto an alkyl group. Similarly, the term “alkthioalkenyls” refers tomoieties wherein an alkyl, alkenyl or alkynyl group is bonded to asulfur atom which is covalently bonded to an alkenyl group; andalkthioalkynyls” refers to moieties wherein an alkyl, alkenyl or alkynylgroup is bonded to a sulfur atom which is covalently bonded to analkynyl group.

As used herein, “amine” or “amino” refers to —NH₂. “Alkylamino” includesgroups of compounds wherein the nitrogen of —NH₂ is bound to at leastone alkyl group. Examples of alkylamino groups include benzylamino,methylamino, ethylamino, phenethylamino, etc. “Dialkylamino” includesgroups wherein the nitrogen of —NH₂ is bound to two alkyl groups.Examples of dialkylamino groups include, but are not limited to,dimethylamino and diethylamine. “Acylamino” and “diarylamino” includegroups wherein the nitrogen is bound to at least one or two aryl groups,respectively. “Aminoacyl” and “aminoaryloxy” refer to aryl and aryloxysubstituted with amino. “Alkylarylamino,” “alkylaminoaryl” or“arylaminoalkyl” refers to an amino group which is bound to at least onealkyl group and at least one aryl group. “Alkaminoalkyl” refers to analkyl, alkenyl, or alkynyl group bound to a nitrogen atom which is alsobound to an alkyl group. “Acylamino” includes groups wherein nitrogen isbound to an acyl group. Examples of acylamino include, but are notlimited to, alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureidogroups.

The term “amide” or “aminocarboxy” includes compounds or moieties thatcontain a nitrogen atom that is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups thatinclude alkyl, alkenyl or alkynyl groups bound to an amino group whichis bound to the carbon of a carbonyl or thiocarbonyl group. It alsoincludes “arylaminocarboxy” groups that include aryl or heteroarylmoieties bound to an amino group that is bound to the carbon of acarbonyl or thiocarbonyl group. The terms “alkylaminocarboxy”,“alkenylaminocarboxy”, “alkynylaminocarboxy” and “arylaminocarboxy”include moieties wherein alkyl, alkenyl, alkynyl and aryl moieties,respectively, are bound to a nitrogen atom which is in turn bound to thecarbon of a carbonyl group. Amides can be substituted with substituentssuch as straight chain alkyl, branched alkyl, cycloalkyl, aryl,heteroaryl or heterocycle. Substituents on amide groups may be furthersubstituted.

The term “amine protecting group” refers to a protecting group foramines. Examples of amine protecting groups include but are not limitedto fluorenylmethyloxycarbonyl (“Fmoc”), carboxybenzyl (“Cbz”),tert-butyloxycarbonyl (“BOC”), dimethoxybenzyl (“DMB”), acetyl (“Ac”),trifluoroacetyl, phthalimide, benzyl (“Bn”), Trityl (triphenylmethyl,Tr), benzylideneamine, Tosyl (Ts). See also Chem. Rev. 2009, 109,2455-2504 for additional amine protecting groups, the contents of whichare incorporated herein by reference in its entirety.

Compounds of the present disclosure that contain nitrogens can beconverted to N-oxides by treatment with an oxidizing agent (e.g.,3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to affordother compounds of the present disclosure. Thus, all shown and claimednitrogen-containing compounds are considered, when allowed by valencyand structure, to include both the compound as shown and its N-oxidederivative (which can be designated as N→O or

N⁺—O⁻). Furthermore, in other instances, the nitrogens in the compoundsof the present disclosure can be converted to N-hydroxy or N-alkoxycompounds. For example, N-hydroxy compounds can be prepared by oxidationof the parent amine by an oxidizing agent such as m-CPBA. All shown andclaimed nitrogen-containing compounds are also considered, when allowedby valency and structure, to cover both the compound as shown and itsN-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R issubstituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl,3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent disclosure includes all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like, it being understood that not all isomers mayhave the same level of activity. In addition, a crystal polymorphism maybe present for the compounds represented by the formula. It is notedthat any crystal form, crystal form mixture, or anhydride or hydratethereof is included in the scope of the present disclosure.

“Isomerism” means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed “stereoisomers.” Stereoisomers that are notmirror images of one another are termed “diastereoisomers,” andstereoisomers that are non-superimposable mirror images of each otherare termed “enantiomers” or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture.”

A carbon atom bonded to four nonidentical substituents is termed a“chiral center.”

“Chiral isomer” means a compound with at least one chiral center.Compounds with more than one chiral center may exist either as anindividual diastereomer or as a mixture of diastereomers, termed“diastereomeric mixture.” When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahnet al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem.Educ. 1964, 41, 116).

“Geometric isomer” means the diastereomers that owe their existence tohindered rotation about double bonds or a cycloalkyl linker (e.g.,1,3-cylcobutyl). These configurations are differentiated in their namesby the prefixes cis and trans, or Z and E, which indicate that thegroups are on the same or opposite side of the double bond in themolecule according to the Cahn-Ingold-Prelog rules.

It is to be understood that the compounds of the present disclosure maybe depicted as different chiral isomers or geometric isomers. It shouldalso be understood that when compounds have chiral isomeric or geometricisomeric forms, all isomeric forms are intended to be included in thescope of the present disclosure, and the naming of the compounds doesnot exclude any isomeric forms, it being understood that not all isomersmay have the same level of activity.

Furthermore, the structures and other compounds discussed in thisdisclosure include all atropic isomers thereof, it being understood thatnot all atropic isomers may have the same level of activity. “Atropicisomers” are a type of stereoisomer in which the atoms of two isomersare arranged differently in space. Atropic isomers owe their existenceto a restricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solutions wheretautomerization is possible, a chemical equilibrium of the tautomerswill be reached. The exact ratio of the tautomers depends on severalfactors, including temperature, solvent and pH. The concept of tautomersthat are interconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g., innucleobases such as guanine, thymine and cytosine), imine-enamine andenamine-enamine. Examples of lactam-lactim tautomerism are as shownbelow.

It is to be understood that the compounds of the present disclosure maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present disclosure, and the naming ofthe compounds does not exclude any tautomer form. It will be understoodthat certain tautomers may have a higher level of activity than others.

The term “crystal polymorphs”, “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or a salt or solvate thereof)can crystallize in different crystal packing arrangements, all of whichhave the same elemental composition. Different crystal forms usuallyhave different X-ray diffraction patterns, infrared spectral, meltingpoints, density hardness, crystal shape, optical and electricalproperties, stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

The compounds of any formula described herein include the compoundsthemselves, as well as their salts, and their solvates, if applicable.

A salt, for example, can be formed between an anion and a positivelycharged group (e.g., amino) on a compound or a polynucleotide (e.g.,mRNA) disclosed herein. Suitable anions include chloride, bromide,iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate,methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate,malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate,lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate).Suitable anions include pharmaceutically acceptable anions. The term“pharmaceutically acceptable anion” refers to an anion suitable forforming a pharmaceutically acceptable salt. Likewise, a salt can also beformed between a cation and a negatively charged group (e.g.,carboxylate) on a compound or a polynucleotide (e.g., mRNA) disclosedherein. Suitable cations include sodium ion, potassium ion, magnesiumion, calcium ion, and an ammonium cation such as tetramethylammoniumion. The compounds and polynucleotides (e.g., mRNA) disclosed herein mayalso include those salts containing quaternary nitrogen atoms.

Additionally, the compounds of the present disclosure, for example, thesalts of the compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include monohydrates, dihydrates, etc. Nonlimitingexamples of solvates include ethanol solvates, acetone solvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non-stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

As defined herein, the term “derivative” refers to compounds that have acommon core structure, and are substituted with various groups asdescribed herein. For example, all of the compounds represented byformula (I) are modified mRNA caps with the ribose group replaced with a6-membered cyclic structure, and have formula (I) as a common core.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres include,but are not limited to, acyl sulfonimides, tetrazoles, sulfonates andphosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176,1996.

The present disclosure is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium, and isotopes of carbon include C-13 and C-14. For example,when a certain variable (e.g., any of R₂₀-R₂₃) in formula (I) is H orhydrogen, it can be either hydrogen or deuterium.

The use of the articles “a”, “an”, and “the” in both the followingdescription and claims are to be construed to cover both the singularand the plural, unless otherwise indicated herein or clearlycontradicted by context. The terms “comprising”, “having”, “being of” asin “being of a chemical formula”, “including”, and “containing” are tobe construed as open terms (i.e., meaning “including but not limitedto”) unless otherwise noted. Additionally whenever “comprising” oranother open-ended term is used in an embodiment, it is to be understoodthat the same embodiment can be more narrowly claimed using theintermediate term “consisting essentially of” or the closed term“consisting of.”

As used herein, the expressions “one or more of A, B, or C,” “one ormore A, B, or C,” “one or more of A, B, and C,” “one or more A, B, andC” and the like are used interchangeably and all refer to a selectionfrom a group consisting of A, B, and/or C, i.e., one or more As, one ormore Bs, one or more Cs, or any combination thereof.

The present disclosure provides methods for the synthesis of thecompounds of any of the formulae described herein. The presentdisclosure also provides detailed methods for the synthesis of variousdisclosed compounds according to the following schemes as shown in theExamples.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the inventionremains operable. Moreover, two or more steps or actions can beconducted simultaneously.

The synthetic processes of the disclosure can tolerate a wide variety offunctional groups, therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt thereof.

Compounds of the present disclosure can be prepared in a variety of waysusing commercially available starting materials, compounds known in theliterature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5th edition, John Wiley & Sons: New York, 2001; Greene,T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis, 3^(rd)edition, John Wiley & Sons: New York, 1999; R. Larock, ComprehensiveOrganic Transformations, VCH Publishers (1989); L. Fieser and M. Fieser,Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons(1994); and L. Paquette, ed., Encyclopedia of Reagents for OrganicSynthesis, John Wiley and Sons (1995), incorporated by reference herein,are useful and recognized reference textbooks of organic synthesis knownto those in the art. The following descriptions of synthetic methods aredesigned to illustrate, but not to limit, general procedures for thepreparation of compounds of the present disclosure.

The compounds of this disclosure having any of the formulae describedherein may be prepared according to the procedures illustrated inSchemes 1-3 below, from commercially available starting materials orstarting materials which can be prepared using literature procedures.The variables (e.g., Y₂) in the scheme are as defined herein for formula(I) unless otherwise specified.

One of ordinary skill in the art will note that, during the reactionsequences and synthetic schemes described herein, the order of certainsteps may be changed, such as the introduction and removal of protectinggroups.

One of ordinary skill in the art will recognize that certain groups mayrequire protection from the reaction conditions via the use ofprotecting groups. Protecting groups may also be used to differentiatesimilar functional groups in molecules. A list of protecting groups andhow to introduce and remove these groups can be found in Greene, T. W.,Wuts, P. G. M., Protective Groups in Organic Synthesis, 3^(rd) edition,John Wiley & Sons: New York, 1999.

Preferred protecting groups include, but are not limited to:

For a hydroxyl moiety: TBS, benzyl, THP, Ac

For carboxylic acids: benzyl ester, methyl ester, ethyl ester, allylester

For amines: Fmoc, Cbz, BOC, DMB, Ac, Bn, Tr, Ts, trifluoroacetyl,phthalimide, benzylideneamine

For diols: Ac (×2) TBS (×2), or when taken together acetonides

For thiols: Ac

For benzimidazoles: SEM, benzyl, PMB, DMB

For aldehydes: di-alkyl acetals such as dimethoxy acetal or diethylacetyl.

In the reaction schemes described herein, multiple stereoisomers may beproduced. When no particular stereoisomer is indicated, it is understoodto mean all possible stereoisomers that could be produced from thereaction. A person of ordinary skill in the art will recognize that thereactions can be optimized to give one isomer preferentially, or newschemes may be devised to produce a single isomer. If mixtures areproduced, techniques such as preparative thin layer chromatography,preparative HPLC, preparative chiral HPLC, or preparative SFC may beused to separate the isomers.

As illustrated in Scheme 1 above, commercially available phosphoramidite(a) is condensed under acidic conditions with the appropriate diolHO—Y₂—OH (e.g., ethylene glycol). The initial ratio ofphosphoramidite-to-diol is equimolar, and the formation of themono-substituted P(III) ester is monitored by LCMS. As the addition isfound to be complete, additional 1 molar equivalent of phosphoramidite(a) is added. The resulting bis-P(III)-phosphodiester is oxidized withtert-butyl hydroperoxide. Treatment with base, such as diethylamine,induces a β-elimination of the cyanoethyl groups to yield thebis-phosphate ester (b). Treatment with a nucleophilic base, such asmethylamine, induces removal of the amide protecting groups to yield (c)and this is followed by fluoride-mediated 2′-O-de-silylation. Acidtreatment (TFA) completes the global deprotection and the finalbis-N-7-methylation afforded the final compound (d).

Scheme 2 above illustrates an alternative approach to synthesizing thecompounds described herein. According to this, guanosine (aa) isconverted to the labile 2′-3′-phenylboronate (bb), which is condensedwith the bis-phosphoramidite (ee). The primary adduct (ft) is oxidizedto the respective phosphotriester (gg), and the protecting groups aresequentially removed. The compound can be purified by ion-exchangechromatography and a symmetrical N7-methylation produces Compound 3.

Scheme 3 above illustrates an approach to synthesizing the compoundsdescribed herein. Phosphoramidite (aaa) and bis(2-cyanoethyl) phosphate(bbb) are coupled to form(bis(2-cyanoethoxy)phosphoryl)oxy)-hydroxypropyl(cyanoethyl)phosphate(ccc), which is then coupled with another 1 molar equivalent ofphosphoramidite (aaa) to yield the primary adduct (ddd). A symmetricalN7-methylation of ddd produces Compound 7. The compound can be purifiedby reverse phase chromatography.

A person of ordinary skill in the art will recognize that in the aboveschemes the order of certain steps may be interchangeable.

Cap analogs described herein are used for the synthesis of 5′ capped RNAmolecules in in vitro transcription reactions. Substitution of capanalog for a portion of the GTP in a transcription reaction results inthe incorporation of the cap structure into a corresponding fraction ofthe transcripts. Capped mRNAs are generally translated more efficientlyin reticulocyte lysate and wheat germ in vitro translation systems. Itis important that in vitro transcripts be capped for microinjectionexperiments because uncapped mRNAs are rapidly degraded. Cap analogs arealso used as a highly specific inhibitor of the initiation step ofprotein synthesis.

Accordingly, in another aspect, the present disclosure provides methodsof synthesizing an RNA molecule in vitro. The method can includereacting unmodified or modified ATP, unmodified or modified CTP,unmodified or modified UTP, unmodified or modified GTP, a compound offormula (I) or a stereoisomer, tautomer or salt thereof, and apolynucleotide template; in the presence an RNA polymerase; under acondition conducive to transcription by the RNA polymerase of thepolynucleotide template into one or more RNA copies; whereby at leastsome of the RNA copies incorporate the compound of formula (I) or astereoisomer, tautomer or salt thereof to make an RNA molecule.

Also provided herein is a kit for capping an RNA transcript. The kitincludes a compound of formula (I) and an RNA polymerase. The kit mayalso include one or more of nucleotides, ribonuclease inhibitor, anenzyme buffer, and an nucleotide buffer.

In another aspect, the RNA molecule may be cappedpost-transcriptionally. For example, recombinant vaccinia virus cappingenzyme and recombinant 2′-O-methyltransferase enzyme can create acanonical 5′-5′-triphosphate linkage between the 5′-terminal nucleotideof an mRNA and a guanine cap nucleotide wherein the cap guanine containsan N7 methylation and the 5′-terminal nucleotide of the mRNA contains a2′-O-methyl.

In yet another aspect, the present disclosure provides an RNA molecule(e.g., mRNA) whose 5′ end comprises a compound (e.g., a cap analog)disclosed herein. For example, the 5′ end of the RNA molecule comprisesa compound of formula (III), (IIIa) or (IIIb):

wherein the wavy line indicates the attachment point to the rest of theRNA molecule.

In embodiments, the variables in formulae (III), (IIIa) and (IIIb) areas defined herein for formula (I), where applicable.

In embodiments, the RNA molecule is an mRNA molecule.

In embodiments, the RNA molecule is an in vitro transcribed mRNAmolecule (IVT mRNA).

In some embodiments, the RNA and mRNA of the disclosure, except for the5′ end cap thereof, is an unmodified RNA or mRNA molecule which has thesame sequence and structure as that of a natural RNA or mRNA molecule.In other embodiments, the RNA and mRNA of the disclosure, in addition tothe modifications on the 5′ end cap disclosed herein, may include atleast one chemical modification as described herein.

Generally, the length of the IVT polynucleotide (e.g., IVT mRNA)encoding a polypeptide of interest is greater than about 30 nucleotidesin length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60,70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600,1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000,7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000,70,000, 80,000, 90,000 or up to and including 100,000 nucleotides).

In some embodiments, the IVT polynucleotide (e.g., IVT mRNA) includesfrom about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000,from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000,from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000,from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000,from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to25,000, from 2,000 to 50,000, from 2,000 to 70,000, or from 2,000 to100,000 nucleotides).

In some embodiments, a nucleic acid as described herein is a chimericpolynucleotide. Chimeric polynucleotides, or RNA constructs, maintain amodular organization similar to IVT polynucleotides, but the chimericpolynucleotides comprise one or more structural and/or chemicalmodifications or alterations which impart useful properties to thepolynucleotide. As such, the chimeric polynucleotides which are modifiedmRNA molecules of the present disclosure are termed “chimeric modifiedmRNA” or “chimeric mRNA.” Chimeric polynucleotides have portions orregions which differ in size and/or chemical modification pattern,chemical modification position, chemical modification percent orchemical modification population and combinations of the foregoing.

In embodiments, the RNA and mRNA of the disclosure is a component of amultimeric mRNA complex.

In another aspect, the disclosure also provides a method of producing amultimeric mRNA complex. In some embodiments, a multimeric mRNA complexis formed by a heating and stepwise cooling protocol. For example, amixture of 5 μM of each mRNA desired to be incorporated into themultimeric complex can be placed in a buffer containing 50 mM2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris) pH 7.5, 150 mM sodiumchloride (NaCl), and 1 mM ethylene-diamine-tetra-acetic acid (EDTA). Themixture can be heated to 65° C. for 5 minutes, 60° C. for 5 minutes, 40°C. for 2 minutes, and then cooled to 4° C. for 10 minutes, resulting inthe formation of a multimeric complex.

In embodiments, the RNA and mRNA of the disclosure are substantiallynon-toxic and non-mutagenic.

In some embodiments, the RNA and mRNA of the disclosure, when introducedto a cell, may exhibit reduced degradation in the cell, as compared to anatural polynucleotide.

As described herein, the polynucleotides (e.g., mRNA) of the disclosurepreferably do not substantially induce an innate immune response of acell into which the polynucleotide (e.g., mRNA) is introduced. Featuresof an induced innate immune response include 1) increased expression ofpro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I,MDA5, etc., and/or 3) termination or reduction in protein translation.

In some embodiments, nucleic acids disclosed herein include a firstregion of linked nucleosides encoding a polypeptide of interest (e.g., acoding region), a first flanking region located at the 5′-terminus ofthe first region (e.g., a 5′-UTR), a second flanking region located atthe 3′-terminus of the first region (e.g., a 3′-UTR), at least one5′-cap region, and a 3′-stabilizing region. In some embodiments, anucleic acid or polynucleotide further includes a poly-A region or aKozak sequence (e.g., in the 5′-UTR). In some cases, polynucleotides maycontain one or more intronic nucleotide sequences capable of beingexcised from the polynucleotide. In some embodiments, a polynucleotideor nucleic acid (e.g., an mRNA) may include a 5′ cap structure, a chainterminating nucleotide, a stem loop, a polyA sequence, and/or apolyadenylation signal. In some embodiments, any one of the regions ofthe polynucleotides of the disclosure includes at least one alternativenucleoside. For example, the 3′-stabilizing region may contain analternative nucleoside such as an L-nucleoside, an inverted thymidine,or a 2′-O-methyl nucleoside and/or the coding region, 5′-UTR, 3′-UTR, orcap region may include an alternative nucleoside such as a 5-substituteduridine (e.g., 5-methoxyuridine), a 1-substituted pseudouridine (e.g.,1-methyl-pseudouridine), and/or a 5-substituted cytidine (e.g.,5-methyl-cytidine).

Generally, the shortest length of a polynucleotide can be the length ofthe polynucleotide sequence that is sufficient to encode for adipeptide. In another embodiment, the length of the polynucleotidesequence is sufficient to encode for a tripeptide. In anotherembodiment, the length of the polynucleotide sequence is sufficient toencode for a tetrapeptide. In another embodiment, the length of thepolynucleotide sequence is sufficient to encode for a pentapeptide. Inanother embodiment, the length of the polynucleotide sequence issufficient to encode for a hexapeptide. In another embodiment, thelength of the polynucleotide sequence is sufficient to encode for aheptapeptide. In another embodiment, the length of the polynucleotidesequence is sufficient to encode for an octapeptide. In anotherembodiment, the length of the polynucleotide sequence is sufficient toencode for a nonapeptide. In another embodiment, the length of thepolynucleotide sequence is sufficient to encode for a decapeptide.

Examples of dipeptides that the alternative polynucleotide sequences canencode for include, but are not limited to, carnosine and anserine.

In some cases, a polynucleotide is greater than 30 nucleotides inlength. In another embodiment, the polynucleotide molecule is greaterthan 35 nucleotides in length. In another embodiment, the length is atleast 40 nucleotides. In another embodiment, the length is at least 45nucleotides. In another embodiment, the length is at least 55nucleotides. In another embodiment, the length is at least 50nucleotides. In another embodiment, the length is at least 60nucleotides. In another embodiment, the length is at least 80nucleotides. In another embodiment, the length is at least 90nucleotides. In another embodiment, the length is at least 100nucleotides. In another embodiment, the length is at least 120nucleotides. In another embodiment, the length is at least 140nucleotides. In another embodiment, the length is at least 160nucleotides. In another embodiment, the length is at least 180nucleotides. In another embodiment, the length is at least 200nucleotides. In another embodiment, the length is at least 250nucleotides. In another embodiment, the length is at least 300nucleotides. In another embodiment, the length is at least 350nucleotides. In another embodiment, the length is at least 400nucleotides. In another embodiment, the length is at least 450nucleotides. In another embodiment, the length is at least 500nucleotides. In another embodiment, the length is at least 600nucleotides. In another embodiment, the length is at least 700nucleotides. In another embodiment, the length is at least 800nucleotides. In another embodiment, the length is at least 900nucleotides. In another embodiment, the length is at least 1000nucleotides. In another embodiment, the length is at least 1100nucleotides. In another embodiment, the length is at least 1200nucleotides. In another embodiment, the length is at least 1300nucleotides. In another embodiment, the length is at least 1400nucleotides. In another embodiment, the length is at least 1500nucleotides. In another embodiment, the length is at least 1600nucleotides. In another embodiment, the length is at least 1800nucleotides. In another embodiment, the length is at least 2000nucleotides. In another embodiment, the length is at least 2500nucleotides. In another embodiment, the length is at least 3000nucleotides. In another embodiment, the length is at least 4000nucleotides. In another embodiment, the length is at least 5000nucleotides, or greater than 5000 nucleotides.

Nucleic acids and polynucleotides disclosed herein may include one ormore naturally occurring components, including any of the canonicalnucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), orT (thymidine). In one embodiment, all or substantially of thenucleotides comprising (a) the 5′-UTR, (b) the open reading frame (ORF),(c) the 3′-UTR, (d) the poly A tail, and any combination of (a, b, c ord above) comprise naturally occurring canonical nucleotides A(adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).

Nucleic acids and polynucleotides disclosed herein may include one ormore alternative components (e.g., in a 3′-stabilizing region), asdescribed herein, which impart useful properties including increasedstability and/or the lack of a substantial induction of the innateimmune response of a cell into which the polynucleotide is introduced.For example, a modified (e.g., altered or alternative) polynucleotide ornucleic acid exhibits reduced degradation in a cell into which thepolynucleotide or nucleic acid is introduced, relative to acorresponding unaltered polynucleotide or nucleic acid. Thesealternative species may enhance the efficiency of protein production,intracellular retention of the polynucleotides, and/or viability ofcontacted cells, as well as possess reduced immunogenicity.

Polynucleotides and nucleic acids may be naturally or non-naturallyoccurring. Polynucleotides and nucleic acids may include one or moremodified (e.g., altered or alternative) nucleobases, nucleosides,nucleotides, or combinations thereof. The nucleic acids andpolynucleotides disclosed herein can include any suitable modificationor alteration, such as to the nucleobase, the sugar, or theinternucleoside linkage (e.g., to a linking phosphate/to aphosphodiester linkage/to the phosphodiester backbone). In certainembodiments, alterations (e.g., one or more alterations) are present ineach of the nucleobase, the sugar, and the internucleoside linkage.Alterations according to the present disclosure may be alterations ofribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), e.g., thesubstitution of the 2′-OH of the ribofuranosyl ring to 2′-H, threosenucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids(PNAs), locked nucleic acids (LNAs), or hybrids thereof. Additionalalterations are described herein.

Polynucleotides and nucleic acids may or may not be uniformly alteredalong the entire length of the molecule. For example, one or more or alltypes of nucleotide (e.g., purine or pyrimidine, or any one or more orall of A, G, U, C) may or may not be uniformly altered in apolynucleotide or nucleic acid, or in a given predetermined sequenceregion thereof. In some instances, all nucleotides X in a polynucleotideof the disclosure (or in a given sequence region thereof) are altered,wherein X may any one of nucleotides A, G, U, C, or any one of thecombinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

Different sugar alterations and/or internucleoside linkages (e.g.,backbone structures) may exist at various positions in thepolynucleotide. One of ordinary skill in the art will appreciate thatthe nucleotide analogs or other alteration(s) may be located at anyposition(s) of a polynucleotide such that the function of thepolynucleotide is not substantially decreased. An alteration may also bea 5′- or 3′-terminal alteration. In some embodiments, the polynucleotideincludes an alteration at the 3′-terminus. The polynucleotide maycontain from about 1% to about 100% alternative nucleotides (either inrelation to overall nucleotide content, or in relation to one or moretypes of nucleotide, i.e., any one or more of A, G, U or C) or anyintervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%,from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10%to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%,from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%,from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%,from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%,from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%,and from 95% to 100%). It will be understood that any remainingpercentage is accounted for by the presence of A, G, U, or C.

The polynucleotides may contain at a minimum one and at maximum 100%alternative nucleotides, or any intervening percentage, such as at least5% alternative nucleotides, at least 10% alternative nucleotides, atleast 25% alternative nucleotides, at least 50% alternative nucleotides,at least 80% alternative nucleotides, or at least 90% alternativenucleotides. For example, the polynucleotides may contain an alternativepyrimidine such as an alternative uracil or cytosine. In someembodiments, at least 5%, at least 10%, at least 25%, at least 50%, atleast 80%, at least 90% or 100% of the uracil in the polynucleotide isreplaced with an alternative uracil (e.g., a 5-substituted uracil). Thealternative uracil can be replaced by a compound having a single uniquestructure, or can be replaced by a plurality of compounds havingdifferent structures (e.g., 2, 3, 4 or more unique structures). In someembodiments, at least 5%, at least 10%, at least 25%, at least 50%, atleast 80%, at least 90% or 100% of the cytosine in the polynucleotide isreplaced with an alternative cytosine (e.g., a 5-substituted cytosine).The alternative cytosine can be replaced by a compound having a singleunique structure, or can be replaced by a plurality of compounds havingdifferent structures (e.g., 2, 3, 4 or more unique structures).

In certain embodiments, it may desirable for an RNA molecule (e.g.,mRNA) introduced into the cell to be degraded intracellularly. Forexample, degradation of an RNA molecule may be preferable if precisetiming of protein production is desired. Thus, in some embodiments, thedisclosure provides an RNA molecule containing a degradation domain,which is capable of being acted on in a directed manner within a cell.

The term “polynucleotide,” in its broadest sense, includes any compoundand/or substance that is or can be incorporated into an oligonucleotidechain. Exemplary polynucleotides for use in accordance with the presentdisclosure include, but are not limited to, one or more of DNA, RNAincluding messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents,RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes,catalytic DNA, RNAs that induce triple helix formation, aptamers,vectors, etc., described in detail herein. In some embodiments, thepolynucleotides may include one or more messenger RNAs (mRNAs) havingone or more modified nucleoside or nucleotides (i.e., unnatural mRNAmolecules).

In some embodiments, a nucleic acid (e.g. mRNA) molecule, formula,composition or method associated therewith comprises one or morepolynucleotides comprising features as described in WO2002/098443,WO2003/051401, WO2008/052770, WO2009127230, WO2006122828, WO2008/083949,WO2010088927, WO2010/037539, WO2004/004743, WO2005/016376,WO2006/024518, WO2007/095976, WO2008/014979, WO2008/077592,WO2009/030481, WO2009/095226, WO2011069586, WO2011026641, WO2011/144358,WO2012019780, WO2012013326, WO2012089338, WO2012113513, WO2012116811,WO2012116810, WO2013113502, WO2013113501, WO2013113736, WO2013143698,WO2013143699, WO2013143700, WO2013/120626, WO2013120627, WO2013120628,WO2013120629, WO2013174409, WO2014127917, WO2015/024669, WO2015/024668,WO2015/024667, WO2015/024665, WO2015/024666, WO2015/024664,WO2015101415, WO2015101414, WO2015024667, WO2015062738, WO2015101416,the contents of each of which are incorporated by reference herein.

Nucleobase Alternatives

The alternative nucleosides and nucleotides can include an alternativenucleobase. A nucleobase of a nucleic acid is an organic base such as apurine or pyrimidine or a derivative thereof. A nucleobase may be acanonical base (e.g., adenine, guanine, uracil, thymine, and cytosine).These nucleobases can be altered or wholly replaced to providepolynucleotide molecules having enhanced properties, e.g., increasedstability such as resistance to nucleases. Non-canonical or modifiedbases may include, for example, one or more substitutions ormodifications including but not limited to alkyl, aryl, halo, oxo,hydroxyl, alkyloxy, and/or thio substitutions; one or more fused or openrings; oxidation; and/or reduction.

Alternative nucleotide base pairing encompasses not only the standardadenine-thymine, adenine-uracil, or guanine-cytosine base pairs, butalso base pairs formed between nucleotides and/or alternativenucleotides including non-standard or alternative bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between thealternative nucleotide inosine and adenine, cytosine, or uracil.

In some embodiments, the nucleobase is an alternative uracil. Exemplarynucleobases and nucleosides having an alternative uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uracil,6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s²U), 4-thio-uracil(s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil(ho⁵U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or5-bromo-uracil), 3-methyl-uracil (m³U), 5-methoxy-uracil (mo⁵U), uracil5-oxyacetic acid (cmo⁵U), uracil 5-oxyacetic acid methyl ester (mcmo⁵U),5-carboxymethyl-uracil (cm⁵U), 1-carboxymethyl-pseudouridine,5-carboxyhydroxymethyl-uracil (chm⁵U), 5-carboxyhydroxymethyl-uracilmethyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uracil (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uracil (mcm⁵s²U),5-aminomethyl-2-thio-uracil (nm⁵s²U), 5-methylaminomethyl-uracil(mnm⁵U), 5-methylaminomethyl-2-thio-uracil (mnm⁵s²U),5-methylaminomethyl-2-seleno-uracil(mnm⁵se²U), 5-carbamoylmethyl-uracil(ncm⁵U), 5-carboxymethylaminomethyl-uracil (cmnm⁵U),5-carboxymethylaminomethyl-2-thio-uracil (cmnm⁵s²U), 5-propynyl-uracil,1-propynyl-pseudouracil, 5-taurinomethyl-uracil (τm⁵U),1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uracil(τm⁵s²U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uracil (m⁵U, i.e., havingthe nucleobase deoxythymine), 1-methyl-pseudouridine5-methyl-2-thio-uracil (m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D),dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m⁵D),2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil,2-methoxy-4-thio-uracil, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uracil (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ iv),5-(isopentenylaminomethyl)uracil (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uracil (inm⁵s²U),5,2′-O-dimethyl-uridine (m⁵Um), 2-thio-2′-O methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uracil,deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil,5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thio-uracil,5-carboxymethyl-2-thio-uracil, 5-cyanomethyl-uracil,5-methoxy-2-thio-uracil, and 5-[3-(1-E-propenylamino)]uracil.

In some embodiments, the nucleobase is an alternative cytosine.Exemplary nucleobases and nucleosides having an alternative cytosineinclude 5-aza-cytosine, 6-aza-cytosine, pseudoisocytidine,3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl-cytosine(f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C),5-halo-cytosine (e.g., 5-iodo-cytosine), 5-hydroxymethyl-cytosine(hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytosine,pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C),2-thio-5-methyl-cytosine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytosine, 2-methoxy-5-methyl-cytosine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k2C), 5,2′-O-dimethyl-cytidine (mSCm),N4-acetyl-2′-O-methyl-cytidine (ac4Cm), N4,2′-O-dimethyl-cytidine(m4Cm), 5-formyl-2′-O-methyl-cytidine (f5Cm),N4,N4,2′-O-trimethyl-cytidine (m42Cm), 1-thio-cytosine,5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine, and5-(2-azidoethyl)-cytosine.

In some embodiments, the nucleobase is an alternative adenine. Exemplarynucleobases and nucleosides having an alternative adenine include2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenine (m1A),2-methyl-adenine (m2A), N6-methyl-adenine (m6A),2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A),2-methylthio-N6-isopentenyl-adenine (ms2i6A),N6-(cis-hydroxyisopentenyl)adenine (io6A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A),N6-glycinylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A),N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A),2-methylthio-N6-threonylcarbamoyl-adenine (ms2 g6A),N6,N6-dimethyl-adenine (m62A), N6-hydroxynorvalylcarbamoyl-adenine(hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A),N6-acetyl-adenine (ac6A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, N6,2′-O-dimethyl-adenosine (m6Am),N6,N6,2′-O-trimethyl-adenosine (m62Am), 1,2′-O-dimethyl-adenosine(m1Am), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine,N6-(19-amino-pentaoxanonadecyl)-adenine, 2,8-dimethyl-adenine,N6-formyl-adenine, and N6-hydroxymethyl-adenine.

In some embodiments, the nucleobase is an alternative guanine. Exemplarynucleobases and nucleosides having an alternative guanine includeinosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodifiedhydroxywybutosine (OHyW*), 7-deaza-guanine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanine (preQ0), 7-aminomethyl-7-deaza-guanine(preQ1), archaeosine (G+), 7-deaza-8-aza-guanine, 6-thio-guanine,6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine(m7G), 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine,1-methyl-guanine (m1G), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine(m22G), N2,7-dimethyl-guanine (m2,7G), N2, N2,7-dimethyl-guanine(m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine,1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine,N2,N2-dimethyl-6-thio-guanine, N2-methyl-2′-O-methyl-guanosine (m2Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m22Gm),1-methyl-2′-O-methyl-guanosine (m1Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m2,7Gm), 2′-O-methyl-inosine (Im),1,2′-O-dimethyl-inosine (m1Im), 1-thio-guanine, and O-6-methyl-guanine.

The alternative nucleobase of a nucleotide can be independently apurine, a pyrimidine, a purine or pyrimidine analog. For example, thenucleobase can be an alternative to adenine, cytosine, guanine, uracil,or hypoxanthine. In another embodiment, the nucleobase can also include,for example, naturally-occurring and synthetic derivatives of a base,including pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine,7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; or 1,3,5 triazine. When the nucleotides aredepicted using the shorthand A, G, C, T or U, each letter refers to therepresentative base and/or derivatives thereof, e.g., A includes adenineor adenine analogs, e.g., 7-deaza adenine).

Alterations on the Sugar

Nucleosides include a sugar molecule (e.g., a 5-carbon or 6-carbonsugar, such as pentose, ribose, arabinose, xylose, glucose, galactose,or a deoxy derivative thereof) in combination with a nucleobase, whilenucleotides are nucleosides containing a nucleoside and a phosphategroup or alternative group (e.g., boranophosphate, thiophosphate,selenophosphate, phosphonate, alkyl group, amidate, and glycerol). Anucleoside or nucleotide may be a canonical species, e.g., a nucleosideor nucleotide including a canonical nucleobase, sugar, and, in the caseof nucleotides, a phosphate group, or may be an alternative nucleosideor nucleotide including one or more alternative components. For example,alternative nucleosides and nucleotides can be altered on the sugar ofthe nucleoside or nucleotide. In some embodiments, the alternativenucleosides or nucleotides include the structure:

In each of the Formulae II′, III′, IV′ and V′,

each of m and n is independently, an integer from 0 to 5,

each of U and U′ independently, is O, S, N(RU)_(nu), or C(RU)_(nu),wherein nu is an integer from 0 to 2 and each RU is, independently, H,halo, or optionally substituted alkyl;

each of R^(1′), R^(1″), R^(2′), R^(1″), R^(2″), R¹, R², R³, R⁴, and R⁵is, independently, if present, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, orabsent; wherein the combination of R³ with one or more of R^(1′),R^(1″), R^(2′), R^(2″), or R⁵ (e.g., the combination of R^(1′) and R³,the combination of R^(1″) and R³, the combination of R^(2′) and R³, thecombination of R^(2″) and R³, or the combination of R⁵ and R³) can jointogether to form optionally substituted alkylene or optionallysubstituted heteroalkylene and, taken together with the carbons to whichthey are attached, provide an optionally substituted heterocyclyl (e.g.,a bicyclic, tricyclic, or tetracyclic heterocyclyl); wherein thecombination of R⁵ with one or more of R^(1′), R^(1″), R^(2′), or R^(2″)(e.g., the combination of R^(1′) and R⁵, the combination of R^(1″) andR⁵, the combination of R^(2′) and R⁵, or the combination of R^(2″) andR⁵) can join together to form optionally substituted alkylene oroptionally substituted heteroalkylene and, taken together with thecarbons to which they are attached, provide an optionally substitutedheterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl);and wherein the combination of R⁴ and one or more of R^(1′), R^(1″),R^(2′), R²″, R³, or R⁵ can join together to form optionally substitutedalkylene or optionally substituted heteroalkylene and, taken togetherwith the carbons to which they are attached, provide an optionallysubstituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclicheterocyclyl); each of m′ and m″ is, independently, an integer from 0 to3 (e.g., from 0 to 2, from 0 to 1, from 1 to 3, or from 1 to 2);

each of Y¹, Y², and Y³, is, independently, O, S, Se, optionallysubstituted alkylene, or optionally substituted heteroalkylene, whereinR^(N1) is H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl, orabsent;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene; and

B is a nucleobase, either modified or unmodified. In some embodiments,the 2′-hydroxy group (OH) can be modified or replaced with a number ofdifferent substituents. Exemplary substitutions at the 2′-positioninclude, but are not limited to, H, azido, halo (e.g., fluoro),optionally substituted C₁₋₆ alkyl (e.g., methyl); optionally substitutedC₁₋₆ alkoxy (e.g., methoxy or ethoxy); optionally substituted C₆₋₁₀aryloxy; optionally substituted C₃₋₈ cycloalkyl; optionally substitutedC₆₋₁₀ aryl-C₁₋₆ alkoxy, optionally substituted C₁₋₁₂ (heterocyclyl)oxy;a sugar (e.g., ribose, pentose, or any described herein); apolyethyleneglycol (PEG), —O(CH₂CH₂O)_(n)CH₂CH₂OR, where R is H oroptionally substituted alkyl, and n is an integer from 0 to 20 (e.g.,from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10,from 4 to 16, and from 4 to 20); “locked” nucleic acids (LNA) in whichthe 2′-hydroxy is connected by a C₁₋₆ alkylene or C₁₋₆ heteroalkylenebridge to the 4′-carbon of the same ribose sugar, where exemplarybridges included methylene, propylene, ether, or amino bridges;aminoalkyl, as defined herein; aminoalkoxy, as defined herein; amino asdefined herein; and amino acid, as defined herein.

Generally, RNA includes the sugar group ribose, which is a 5-memberedring having an oxygen. Exemplary, non-limiting alternative nucleotidesinclude replacement of the oxygen in ribose (e.g., with S, Se, oralkylene, such as methylene or ethylene); addition of a double bond(e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ringcontraction of ribose (e.g., to form a 4-membered ring of cyclobutane oroxetane); ring expansion of ribose (e.g., to form a 6- or 7-memberedring having an additional carbon or heteroatom, such as foranhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, andmorpholino (that also has a phosphoramidate backbone)); multicyclicforms (e.g., tricyclo and “unlocked” forms, such as glycol nucleic acid(GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol unitsattached to phosphodiester bonds), threose nucleic acid (TNA, whereribose is replace with α-L-threofuranosyl-(3′→2′)), and peptide nucleicacid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose andphosphodiester backbone).

In some embodiments, the sugar group contains one or more carbons thatpossess the opposite stereochemical configuration of the correspondingcarbon in ribose. Thus, a polynucleotide molecule can includenucleotides containing, e.g., arabinose or L-ribose, as the sugar.

In some embodiments, the polynucleotide of the disclosure includes atleast one nucleoside wherein the sugar is L-ribose, 2′-O-methyl-ribose,2′-fluoro-ribose, arabinose, hexitol, an LNA, or a PNA.

Alterations on the Internucleoside Linkage

Alternative nucleotides can be altered on the internucleoside linkage(e.g., phosphate backbone). Herein, in the context of the polynucleotidebackbone, the phrases “phosphate” and “phosphodiester” are usedinterchangeably. Backbone phosphate groups can be altered by replacingone or more of the oxygen atoms with a different substituent.

The alternative nucleotides can include the wholesale replacement of anunaltered phosphate moiety with another internucleoside linkage asdescribed herein. Examples of alternative phosphate groups include, butare not limited to, phosphorothioate, phosphoroselenates,boranophosphates, boranophosphate esters, hydrogen phosphonates,phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. The phosphate linker can also be altered by thereplacement of a linking oxygen with nitrogen (bridgedphosphoramidates), sulfur (bridged phosphorothioates), and carbon(bridged methylene-phosphonates).

The alternative nucleosides and nucleotides can include the replacementof one or more of the non-bridging oxygens with a borane moiety (BH₃),sulfur (thio), methyl, ethyl, and/or methoxy. As a non-limiting example,two non-bridging oxygens at the same position (e.g., the alpha (a), beta03) or gamma (γ) position) can be replaced with a sulfur (thio) and amethoxy.

The replacement of one or more of the oxygen atoms at the α position ofthe phosphate moiety (e.g., α-thio phosphate) is provided to conferstability (such as against exonucleases and endonucleases) to RNA andDNA through the unnatural phosphorothioate backbone linkages.Phosphorothioate DNA and RNA have increased nuclease resistance andsubsequently a longer half-life in a cellular environment.

Other internucleoside linkages that may be employed according to thepresent disclosure, including internucleoside linkages which do notcontain a phosphorous atom, are described herein.

Internal Ribosome Entry Sites

Polynucleotides may contain an internal ribosome entry site (IRES). AnIRES may act as the sole ribosome binding site, or may serve as one ofmultiple ribosome binding sites of an mRNA. A polynucleotide containingmore than one functional ribosome binding site may encode severalpeptides or polypeptides that are translated independently by theribosomes (e.g., multicistronic mRNA). When polynucleotides are providedwith an IRES, further optionally provided is a second translatableregion. Examples of IRES sequences that can be used according to thepresent disclosure include without limitation, those from picornaviruses(e.g., FMDV), pest viruses (CFFV), polio viruses (PV),encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses(FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV),murine leukemia virus (MLV), simian immune deficiency viruses (SIV) orcricket paralysis viruses (CrPV).

5′-UTRs

A 5′-UTR may be provided as a flanking region to polynucleotides (e.g.,mRNAs). A 5′-UTR may be homologous or heterologous to the coding regionfound in a polynucleotide. Multiple 5′-UTRs may be included in theflanking region and may be the same or of different sequences. Anyportion of the flanking regions, including none, may be codon optimizedand any may independently contain one or more different structural orchemical alterations, before and/or after codon optimization.

Shown in Table 21 in U.S. Provisional Application No. 61/775,509, and inTable 21 and in Table 22 in U.S. Provisional Application No. 61/829,372,of which are incorporated herein by reference, is a listing of the startand stop site of alternative polynucleotides (e.g., mRNA) of thedisclosure. In Table 21 each 5′-UTR (5′-UTR-005 to 5′-UTR 68511) isidentified by its start and stop site relative to its native or wildtype (homologous) transcript (ENST; the identifier used in the ENSEMBLdatabase).

To alter one or more properties of a polynucleotide (e.g., mRNA),5′-UTRs which are heterologous to the coding region of an alternativepolynucleotide (e.g., mRNA) may be engineered. The polynucleotides(e.g., mRNA) may then be administered to cells, tissue or organisms andoutcomes such as protein level, localization, and/or half-life may bemeasured to evaluate the beneficial effects the heterologous 5′-UTR mayhave on the alternative polynucleotides (mRNA). Variants of the 5′-UTRsmay be utilized wherein one or more nucleotides are added or removed tothe termini, including A, T, C or G. 5′-UTRs may also becodon-optimized, or altered in any manner described herein.

5′-UTRs, 3′-UTRs, and Translation Enhancer Elements (TEEs)

The 5′-UTR of a polynucleotides (e.g., mRNA) may include at least onetranslation enhancer element. The term “translational enhancer element”refers to sequences that increase the amount of polypeptide or proteinproduced from a polynucleotide. As a non-limiting example, the TEE maybe located between the transcription promoter and the start codon. Thepolynucleotides (e.g., mRNA) with at least one TEE in the 5′-UTR mayinclude a cap at the 5′-UTR. Further, at least one TEE may be located inthe 5′-UTR of polynucleotides (e.g., mRNA) undergoing cap-dependent orcap-independent translation.

In one aspect, TEEs are conserved elements in the UTR which can promotetranslational activity of a polynucleotide such as, but not limited to,cap-dependent or cap-independent translation. The conservation of thesesequences has been previously shown by Panek et al. (Nucleic AcidsResearch, 2013, 1-10) across 14 species including humans.

In one non-limiting example, the TEEs known may be in the 5′-leader ofthe Gtx homeodomain protein (Chappell et al., Proc. Natl. Acad. Sci. USA101:9590-9594, 2004, the TEEs of which are incorporated herein byreference).

In another non-limiting example, TEEs are disclosed as SEQ ID NOs: 1-35in US Patent Publication No. 2009/0226470, SEQ ID NOs: 1-35 in US PatentPublication No. 2013/0177581, SEQ ID NOs: 1-35 in International PatentPublication No. WO2009/075886, SEQ ID NOs: 1-5, and 7-645 inInternational Patent Publication No. WO2012/009644, SEQ ID NO: 1 inInternational Patent Publication No. WO1999/024595, SEQ ID NO: 1 in U.S.Pat. No. 6,310,197, and SEQ ID NO: 1 in U.S. Pat. No. 6,849,405, the TEEsequences of each of which are incorporated herein by reference.

In yet another non-limiting example, the TEE may be an internal ribosomeentry site (IRES), HCV-IRES or an IRES element such as, but not limitedto, those described in U.S. Pat. No. 7,468,275, US Patent PublicationNos. 2007/0048776 and 2011/0124100 and International Patent PublicationNos. WO2007/025008 and WO2001/055369, the IRES sequences of each ofwhich are incorporated herein by reference. The IRES elements mayinclude, but are not limited to, the Gtx sequences (e.g., Gtx9-nt,Gtx8-nt, Gtx7-nt) described by Chappell et al. (Proc. Natl. Acad. Sci.USA 101:9590-9594, 2004) and Zhou et al. (PNAS 102:6273-6278, 2005) andin US Patent Publication Nos. 2007/0048776 and 2011/0124100 andInternational Patent Publication No. WO2007/025008, the IRES sequencesof each of which are incorporated herein by reference.

“Translational enhancer polynucleotides” are polynucleotides whichinclude one or more of the specific TEE exemplified herein and/ordisclosed in the art (see e.g., U.S. Pat. Nos. 6,310,197, 6,849,405,7,456,273, 7,183,395, U.S. Patent Publication Nos. 20090/226470,2007/0048776, 2011/0124100, 2009/0093049, 2013/0177581, InternationalPatent Publication Nos. WO2009/075886, WO2007/025008, WO2012/009644,WO2001/055371 WO1999/024595, and European Patent Nos. 2610341 and2610340; the TEE sequences of each of which are incorporated herein byreference) or their variants, homologs or functional derivatives. One ormultiple copies of a specific TEE can be present in a polynucleotide(e.g., mRNA). The TEEs in the translational enhancer polynucleotides canbe organized in one or more sequence segments. A sequence segment canharbor one or more of the specific TEEs exemplified herein, with eachTEE being present in one or more copies. When multiple sequence segmentsare present in a translational enhancer polynucleotide, they can behomogenous or heterogeneous. Thus, the multiple sequence segments in atranslational enhancer polynucleotide can harbor identical or differenttypes of the specific TEEs exemplified herein, identical or differentnumber of copies of each of the specific TEEs, and/or identical ordifferent organization of the TEEs within each sequence segment.

A polynucleotide (e.g., mRNA) may include at least one TEE that isdescribed in International Patent Publication Nos. WO1999/024595,WO2012/009644, WO2009/075886, WO2007/025008, WO1999/024595, EuropeanPatent Publication Nos. 2610341 and 2610340, U.S. Pat. Nos. 6,310,197,6,849,405, 7,456,273, 7,183,395, and US Patent Publication Nos.2009/0226470, 2011/0124100, 2007/0048776, 2009/0093049, and 2013/0177581the TEE sequences of each of which are incorporated herein by reference.The TEE may be located in the 5′-UTR of the polynucleotides (e.g.,mRNA).

A polynucleotide (e.g., mRNA) may include at least one TEE that has atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% or atleast 99% identity with the TEEs described in US Patent Publication Nos.2009/0226470, 2007/0048776, 2013/0177581 and 2011/0124100, InternationalPatent Publication Nos. WO1999/024595, WO2012/009644, WO2009/075886 andWO2007/025008, European Patent Publication Nos. 2610341 and 2610340,U.S. Pat. Nos. 6,310,197, 6,849,405, 7,456,273, 7,183,395, the TEEsequences of each of which are incorporated herein by reference.

The 5′-UTR of a polynucleotide (e.g., mRNA) may include at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18 at least19, at least 20, at least 21, at least 22, at least 23, at least 24, atleast 25, at least 30, at least 35, at least 40, at least 45, at least50, at least 55 or more than 60 TEE sequences. The TEE sequences in the5′-UTR of a polynucleotide (e.g., mRNA) may be the same or different TEEsequences. The TEE sequences may be in a pattern such as ABABAB,AABBAABBAABB, or ABCABCABC, or variants thereof, repeated once, twice,or more than three times. In these patterns, each letter, A, B, or Crepresent a different TEE sequence at the nucleotide level.

In some cases, the 5′-UTR may include a spacer to separate two TEEsequences. As a non-limiting example, the spacer may be a 15 nucleotidespacer and/or other spacers known in the art. As another non-limitingexample, the 5′-UTR may include a TEE sequence-spacer module repeated atleast once, at least twice, at least 3 times, at least 4 times, at least5 times, at least 6 times, at least 7 times, at least 8 times, at least9 times, or more than 9 times in the 5′-UTR.

In other instances, the spacer separating two TEE sequences may includeother sequences known in the art which may regulate the translation ofthe polynucleotides (e.g., mRNA) of the present disclosure, such as, butnot limited to, miR sequences (e.g., miR binding sites and miR seeds).As a non-limiting example, each spacer used to separate two TEEsequences may include a different miR sequence or component of a miRsequence (e.g., miR seed sequence).

In some instances, the TEE in the 5′-UTR of a polynucleotide (e.g.,mRNA) may include at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 99% or more than 99% of the TEE sequences disclosed in US PatentPublication Nos. 2009/0226470, 2007/0048776, 2013/0177581 and2011/0124100, International Patent Publication Nos. WO1999/024595,WO2012/009644, WO2009/075886 and WO2007/025008, European PatentPublication Nos. 2610341 and 2610340, and U.S. Pat. Nos. 6,310,197,6,849,405, 7,456,273, and 7,183,395 the TEE sequences of each of whichare incorporated herein by reference. In another embodiment, the TEE inthe 5′-UTR of the polynucleotides (e.g., mRNA) of the present disclosuremay include a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a5-20 nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotidefragment of the TEE sequences disclosed in US Patent Publication Nos.2009/0226470, 2007/0048776, 2013/0177581 and 2011/0124100, InternationalPatent Publication Nos. WO1999/024595, WO2012/009644, WO2009/075886 andWO2007/025008, European Patent Publication Nos. 2610341 and 2610340, andU.S. Pat. Nos. 6,310,197, 6,849,405, 7,456,273, and 7,183,395; the TEEsequences of each of which are incorporated herein by reference.

In certain cases, the TEE in the 5′-UTR of the polynucleotides (e.g.,mRNA) of the present disclosure may include at least 5%, at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 99% or more than 99% of the TEEsequences disclosed in Chappell et al. (Proc. Natl. Acad. Sci. USA101:9590-9594, 2004) and Zhou et al. (PNAS 102:6273-6278, 2005), inSupplemental Table 1 and in Supplemental Table 2 disclosed by Wellensieket al (Genome-wide profiling of human cap-independenttranslation-enhancing elements, Nature Methods, 2013;DOI:10.1038/NMETH.2522); the TEE sequences of each of which are hereinincorporated by reference. In another embodiment, the TEE in the 5′-UTRof the polynucleotides (e.g., mRNA) of the present disclosure mayinclude a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a 5-20nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotidefragment of the TEE sequences disclosed in Chappell et al. (Proc. Natl.Acad. Sci. USA 101:9590-9594, 2004) and Zhou et al. (PNAS 102:6273-6278,2005), in Supplemental Table 1 and in Supplemental Table 2 disclosed byWellensiek et al (Genome-wide profiling of human cap-independenttranslation-enhancing elements, Nature Methods, 2013;DOI:10.1038/NMETH.2522); the TEE sequences of each of which isincorporated herein by reference.

In some cases, the TEE used in the 5′-UTR of a polynucleotide (e.g.,mRNA) is an IRES sequence such as, but not limited to, those describedin U.S. Pat. No. 7,468,275 and International Patent Publication No.WO2001/055369, the TEE sequences of each of which are incorporatedherein by reference.

In some instances, the TEEs used in the 5′-UTR of a polynucleotide(e.g., mRNA) may be identified by the methods described in US PatentPublication Nos. 2007/0048776 and 2011/0124100 and International PatentPublication Nos. WO2007/025008 and WO2012/009644, the methods of each ofwhich are incorporated herein by reference.

In some cases, the TEEs used in the 5′-UTR of a polynucleotide (e.g.,mRNA) of the present disclosure may be a transcription regulatoryelement described in U.S. Pat. Nos. 7,456,273 and 7,183,395, US PatentPublication No. 2009/0093049, and International Publication No.WO2001/055371, the TEE sequences of each of which are incorporatedherein by reference. The transcription regulatory elements may beidentified by methods known in the art, such as, but not limited to, themethods described in U.S. Pat. Nos. 7,456,273 and 7,183,395, US PatentPublication No. 2009/0093049, and International Publication No.WO2001/055371, the methods of each of which are incorporated herein byreference.

In yet other instances, the TEE used in the 5′-UTR of a polynucleotide(e.g., mRNA) is a polynucleotide or portion thereof as described in U.S.Pat. Nos. 7,456,273 and 7,183,395, US Patent Publication No.2009/0093049, and International Publication No. WO2001/055371, the TEEsequences of each of which are incorporated herein by reference.

The 5′-UTR including at least one TEE described herein may beincorporated in a monocistronic sequence such as, but not limited to, avector system or a polynucleotide vector. As a non-limiting example, thevector systems and polynucleotide vectors may include those described inU.S. Pat. Nos. 7,456,273 and 7,183,395, US Patent Publication Nos.2007/0048776, 2009/0093049 and 2011/0124100, and International PatentPublication Nos. WO2007/025008 and WO2001/055371, the TEE sequences ofeach of which are incorporated herein by reference.

The TEEs described herein may be located in the 5′-UTR and/or the 3′-UTRof the polynucleotides (e.g., mRNA). The TEEs located in the 3′-UTR maybe the same and/or different than the TEEs located in and/or describedfor incorporation in the 5′-UTR.

In some cases, the 3′-UTR of a polynucleotide (e.g., mRNA) may includeat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18 at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, at least 30, at least 35, at least 40, atleast 45, at least 50, at least 55 or more than 60 TEE sequences. TheTEE sequences in the 3′-UTR of the polynucleotides (e.g., mRNA) of thepresent disclosure may be the same or different TEE sequences. The TEEsequences may be in a pattern such as ABABAB, AABBAABBAABB, orABCABCABC, or variants thereof, repeated once, twice, or more than threetimes. In these patterns, each letter, A, B, or C represent a differentTEE sequence at the nucleotide level.

In one instance, the 3′-UTR may include a spacer to separate two TEEsequences. As a non-limiting example, the spacer may be a 15 nucleotidespacer and/or other spacers known in the art. As another non-limitingexample, the 3′-UTR may include a TEE sequence-spacer module repeated atleast once, at least twice, at least 3 times, at least 4 times, at least5 times, at least 6 times, at least 7 times, at least 8 times, at least9 times, or more than 9 times in the 3′-UTR.

In other cases, the spacer separating two TEE sequences may includeother sequences known in the art which may regulate the translation ofthe polynucleotides (e.g., mRNA) of the present disclosure such as, butnot limited to, miR sequences described herein (e.g., miR binding sitesand miR seeds). As a non-limiting example, each spacer used to separatetwo TEE sequences may include a different miR sequence or component of amiR sequence (e.g., miR seed sequence).

In yet other cases, the incorporation of a miR sequence and/or a TEEsequence changes the shape of the stem loop region which may increaseand/or decrease translation. (see e.g, Kedde et al. A Pumilio-inducedRNA structure switch in p27-3′UTR controls miR-221 and miR-22accessibility. Nature Cell Biology. 2010).

Stem Loops

Polynucleotides (e.g., mRNAs) may include a stem loop such as, but notlimited to, a histone stem loop. The stem loop may be a nucleotidesequence that is about 25 or about 26 nucleotides in length such as, butnot limited to, SEQ ID NOs: 7-17 as described in International PatentPublication No. WO2013/103659, of which SEQ ID NOs: 7-17 areincorporated herein by reference. The histone stem loop may be located3′-relative to the coding region (e.g., at the 3′-terminus of the codingregion). As a non-limiting example, the stem loop may be located at the3′-end of a polynucleotide described herein. In some cases, apolynucleotide (e.g., an mRNA) includes more than one stem loop (e.g.,two stem loops). Examples of stem loop sequences are described inInternational Patent Publication Nos. WO2012/019780 and WO201502667, thestem loop sequences of which are herein incorporated by reference. Insome instances, a polynucleotide includes the stem loop sequenceCAAAGGCTCTTTTCAGAGCCACCA (SEQ ID NO: 1). In others, a polynucleotideincludes the stem loop sequence CAAAGGCUCUUUUCAGAGCCACCA (SEQ ID NO: 2).

A stem loop may be located in a second terminal region of apolynucleotide. As a non-limiting example, the stem loop may be locatedwithin an untranslated region (e.g., 3′-UTR) in a second terminalregion.

In some cases, a polynucleotide such as, but not limited to mRNA, whichincludes the histone stem loop may be stabilized by the addition of a3′-stabilizing region (e.g., a 3′-stabilizing region including at leastone chain terminating nucleoside). Not wishing to be bound by theory,the addition of at least one chain terminating nucleoside may slow thedegradation of a polynucleotide and thus can increase the half-life ofthe polynucleotide.

In other cases, a polynucleotide such as, but not limited to mRNA, whichincludes the histone stem loop may be stabilized by an alteration to the3′-region of the polynucleotide that can prevent and/or inhibit theaddition of oligio(U) (see e.g., International Patent Publication No.WO2013/103659,).

In yet other cases, a polynucleotide such as, but not limited to mRNA,which includes the histone stem loop may be stabilized by the additionof an oligonucleotide that terminates in a 3′-deoxynucleoside,2′,3′-dideoxynucleoside 3′-O-methylnucleosides, 3′-O-ethylnucleosides,3′-arabinosides, and other alternative nucleosides known in the artand/or described herein.

In some instances, the polynucleotides of the present disclosure mayinclude a histone stem loop, a poly-A region, and/or a 5′-cap structure.The histone stem loop may be before and/or after the poly-A region. Thepolynucleotides including the histone stem loop and a poly-A regionsequence may include a chain terminating nucleoside described herein.

In other instances, the polynucleotides of the present disclosure mayinclude a histone stem loop and a 5′-cap structure. The 5′-cap structuremay include, but is not limited to, those described herein and/or knownin the art.

In some cases, the conserved stem loop region may include a miR sequencedescribed herein. As a non-limiting example, the stem loop region mayinclude the seed sequence of a miR sequence described herein. In anothernon-limiting example, the stem loop region may include a miR-122 seedsequence.

In certain instances, the conserved stem loop region may include a miRsequence described herein and may also include a TEE sequence.

In some cases, the incorporation of a miR sequence and/or a TEE sequencechanges the shape of the stem loop region which may increase and/ordecrease translation. (see e.g, Kedde et al. A Pumilio-induced RNAstructure switch in p27-3′UTR controls miR-221 and miR-22 accessibility.Nature Cell Biology. 2010, herein incorporated by reference in itsentirety).

Polynucleotides may include at least one histone stem-loop and a poly-Aregion or polyadenylation signal. Non-limiting examples ofpolynucleotide sequences encoding for at least one histone stem-loop anda poly-A region or a polyadenylation signal are described inInternational Patent Publication No. WO2013/120497, WO2013/120629,WO2013/120500, WO2013/120627, WO2013/120498, WO2013/120626,WO2013/120499 and WO2013/120628, the sequences of each of which areincorporated herein by reference. In certain cases, the polynucleotideencoding for a histone stem loop and a poly-A region or apolyadenylation signal may code for a pathogen antigen or fragmentthereof such as the polynucleotide sequences described in InternationalPatent Publication No WO2013/120499 and WO2013/120628, the sequences ofboth of which are incorporated herein by reference. In other cases, thepolynucleotide encoding for a histone stem loop and a poly-A region or apolyadenylation signal may code for a therapeutic protein such as thepolynucleotide sequences described in International Patent PublicationNo WO2013/120497 and WO2013/120629, the sequences of both of which areincorporated herein by reference. In some cases, the polynucleotideencoding for a histone stem loop and a poly-A region or apolyadenylation signal may code for a tumor antigen or fragment thereofsuch as the polynucleotide sequences described in International PatentPublication No WO2013/120500 and WO2013/120627, the sequences of both ofwhich are incorporated herein by reference. In other cases, thepolynucleotide encoding for a histone stem loop and a poly-A region or apolyadenylation signal may code for a allergenic antigen or anautoimmune self-antigen such as the polynucleotide sequences describedin International Patent Publication No WO2013/120498 and WO2013/120626,the sequences of both of which are incorporated herein by reference.

Poly-A Regions

A polynucleotide or nucleic acid (e.g., an mRNA) may include a polyAsequence and/or polyadenylation signal. A polyA sequence may becomprised entirely or mostly of adenine nucleotides or analogs orderivatives thereof. A polyA sequence may be a tail located adjacent toa 3′ untranslated region of a nucleic acid.

During RNA processing, a long chain of adenosine nucleotides (poly-Aregion) is normally added to messenger RNA (mRNA) molecules to increasethe stability of the molecule. Immediately after transcription, the3′-end of the transcript is cleaved to free a 3′-hydroxy. Then poly-Apolymerase adds a chain of adenosine nucleotides to the RNA. Theprocess, called polyadenylation, adds a poly-A region that is between100 and 250 residues long.

Unique poly-A region lengths may provide certain advantages to thealternative polynucleotides of the present disclosure.

Generally, the length of a poly-A region of the present disclosure is atleast 30 nucleotides in length. In another embodiment, the poly-A regionis at least 35 nucleotides in length. In another embodiment, the lengthis at least 40 nucleotides. In another embodiment, the length is atleast 45 nucleotides. In another embodiment, the length is at least 55nucleotides. In another embodiment, the length is at least 60nucleotides. In another embodiment, the length is at least 70nucleotides. In another embodiment, the length is at least 80nucleotides. In another embodiment, the length is at least 90nucleotides. In another embodiment, the length is at least 100nucleotides. In another embodiment, the length is at least 120nucleotides. In another embodiment, the length is at least 140nucleotides. In another embodiment, the length is at least 160nucleotides. In another embodiment, the length is at least 180nucleotides. In another embodiment, the length is at least 200nucleotides. In another embodiment, the length is at least 250nucleotides. In another embodiment, the length is at least 300nucleotides. In another embodiment, the length is at least 350nucleotides. In another embodiment, the length is at least 400nucleotides. In another embodiment, the length is at least 450nucleotides. In another embodiment, the length is at least 500nucleotides. In another embodiment, the length is at least 600nucleotides. In another embodiment, the length is at least 700nucleotides. In another embodiment, the length is at least 800nucleotides. In another embodiment, the length is at least 900nucleotides. In another embodiment, the length is at least 1000nucleotides. In another embodiment, the length is at least 1100nucleotides. In another embodiment, the length is at least 1200nucleotides. In another embodiment, the length is at least 1300nucleotides. In another embodiment, the length is at least 1400nucleotides. In another embodiment, the length is at least 1500nucleotides. In another embodiment, the length is at least 1600nucleotides. In another embodiment, the length is at least 1700nucleotides. In another embodiment, the length is at least 1800nucleotides. In another embodiment, the length is at least 1900nucleotides. In another embodiment, the length is at least 2000nucleotides. In another embodiment, the length is at least 2500nucleotides. In another embodiment, the length is at least 3000nucleotides.

In some instances, the poly-A region may be 80 nucleotides, 120nucleotides, 160 nucleotides in length on an alternative polynucleotidemolecule described herein.

In other instances, the poly-A region may be 20, 40, 80, 100, 120, 140or 160 nucleotides in length on an alternative polynucleotide moleculedescribed herein.

In some cases, the poly-A region is designed relative to the length ofthe overall alternative polynucleotide. This design may be based on thelength of the coding region of the alternative polynucleotide, thelength of a particular feature or region of the alternativepolynucleotide (such as mRNA), or based on the length of the ultimateproduct expressed from the alternative polynucleotide. When relative toany feature of the alternative polynucleotide (e.g., other than the mRNAportion which includes the poly-A region) the poly-A region may be 10,20, 30, 40, 50, 60, 70, 80, 90 or 100% greater in length than theadditional feature. The poly-A region may also be designed as a fractionof the alternative polynucleotide to which it belongs. In this context,the poly-A region may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or moreof the total length of the construct or the total length of theconstruct minus the poly-A region.

In certain cases, engineered binding sites and/or the conjugation ofpolynucleotides (e.g., mRNA) for poly-A binding protein may be used toenhance expression. The engineered binding sites may be sensor sequenceswhich can operate as binding sites for ligands of the localmicroenvironment of the polynucleotides (e.g., mRNA). As a non-limitingexample, the polynucleotides (e.g., mRNA) may include at least oneengineered binding site to alter the binding affinity of poly-A bindingprotein (PABP) and analogs thereof. The incorporation of at least oneengineered binding site may increase the binding affinity of the PABPand analogs thereof.

Additionally, multiple distinct polynucleotides (e.g., mRNA) may belinked together to the PABP (poly-A binding protein) through the 3′-endusing alternative nucleotides at the 3′-terminus of the poly-A region.Transfection experiments can be conducted in relevant cell lines at andprotein production can be assayed by ELISA at 12 hours, 24 hours, 48hours, 72 hours, and day 7 post-transfection. As a non-limiting example,the transfection experiments may be used to evaluate the effect on PABPor analogs thereof binding affinity as a result of the addition of atleast one engineered binding site.

In certain cases, a poly-A region may be used to modulate translationinitiation. While not wishing to be bound by theory, the poly-A regionrecruits PABP which in turn can interact with translation initiationcomplex and thus may be essential for protein synthesis.

In some cases, a poly-A region may also be used in the presentdisclosure to protect against 3′-5′-exonuclease digestion.

In some instances, a polynucleotide (e.g., mRNA) may include a polyA-GQuartet. The G-quartet is a cyclic hydrogen bonded array of fourguanosine nucleotides that can be formed by G-rich sequences in both DNAand RNA. In this embodiment, the G-quartet is incorporated at the end ofthe poly-A region. The resultant polynucleotides (e.g., mRNA) may beassayed for stability, protein production and other parameters includinghalf-life at various time points. It has been discovered that thepolyA-G quartet results in protein production equivalent to at least 75%of that seen using a poly-A region of 120 nucleotides alone.

In some cases, a polynucleotide (e.g., mRNA) may include a poly-A regionand may be stabilized by the addition of a 3′-stabilizing region. Thepolynucleotides (e.g., mRNA) with a poly-A region may further include a5′-cap structure.

In other cases, a polynucleotide (e.g., mRNA) may include a poly-A-GQuartet. The polynucleotides (e.g., mRNA) with a poly-A-G Quartet mayfurther include a 5′-cap structure.

In some cases, the 3′-stabilizing region which may be used to stabilizea polynucleotide (e.g., mRNA) including a poly-A region or poly-A-GQuartet may be, but is not limited to, those described in InternationalPatent Publication No. WO2013/103659, the poly-A regions and poly-A-GQuartets of which are incorporated herein by reference. In other cases,the 3′-stabilizing region which may be used with the present disclosureinclude a chain termination nucleoside such as 3′-deoxyadenosine(cordycepin), 3′-deoxyuridine, 3′-deoxycytosine, 3′-deoxyguanosine,3′-deoxythymine, 2′,3′-dideoxynucleosides, such as2′,3′-dideoxyadenosine, 2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine,2′,3′-dideoxyguanosine, 2′,3′-dideoxythymine, a 2′-deoxynucleoside, oran O-methylnucleoside.

In other cases, a polynucleotide such as, but not limited to mRNA, whichincludes a polyA region or a poly-A-G Quartet may be stabilized by analteration to the 3′-region of the polynucleotide that can preventand/or inhibit the addition of oligio(U) (see e.g., International PatentPublication No. WO2013/103659).

In yet other instances, a polynucleotide such as, but not limited tomRNA, which includes a poly-A region or a poly-A-G Quartet may bestabilized by the addition of an oligonucleotide that terminates in a3′-deoxynucleoside, 2′,3′-dideoxynucleoside 3′-O-methylnucleosides,3′-O-ethylnucleosides, 3′-arabinosides, and other alternativenucleosides known in the art and/or described herein.

Chain Terminating Nucleosides

A nucleic acid may include a chain terminating nucleoside. For example,a chain terminating nucleoside may include those nucleosidesdeoxygenated at the 2′ and/or 3′ positions of their sugar group. Suchspecies may include 3′-deoxyadenosine (cordycepin), 3′-deoxyuridine,3′-deoxycytosine, 3′-deoxyguanosine, 3′-deoxythymine, and2′,3′-dideoxynucleosides, such as 2′,3′-dideoxyadenosine,2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine, and2′,3′-dideoxythymine.

The RNAs and multimeric nucleic acid complexes described herein can beused as therapeutic agents or are therapeutic mRNAs. As used herein, theterm “therapeutic mRNA” refers to an mRNA that encodes a therapeuticprotein. Therapeutic proteins mediate a variety of effects in a hostcell or a subject in order to treat a disease or ameliorate the signsand symptoms of a disease. For example, an RNA or a multimeric structuredescribed herein can be administered to an animal or human subject,wherein the RNA is translated in vivo to produce a therapeutic peptidein the subject in need thereof. Accordingly, provided herein arecompositions, methods, kits, and reagents for treatment or prevention ofdisease or conditions in humans and other mammals. The activetherapeutic agents of the present disclosure include RNAs (e.g., mRNAs)disclosed herein, cells containing the mRNAs or polypeptides translatedfrom the mRNAs, polypeptides translated from mRNAs, cells contacted withcells containing mRNAs or polypeptides translated therefrom, tissuescontaining cells containing the mRNAs described herein and organscontaining tissues containing cells containing the mRNAs describedherein.

In another aspect, the disclosure provides methods and compositionsuseful for protecting RNAs disclosed herein (e.g., RNA transcripts) fromdegradation (e.g., exonuclease mediated degradation), such as methodsand compositions described in US20150050738A1 and WO2015023975A1, thecontents of each of which are herein incorporated by reference in theirentireties.

In some embodiments, the protected RNAs are present outside of cells. Insome embodiments, the protected RNAs are present in cells. In someembodiments, methods and compositions are provided that are useful forpost-transcriptionally altering protein and/or RNA levels in a targetedmanner. In some embodiments, methods disclosed herein involve reducingor preventing degradation or processing of targeted RNAs therebyelevating steady state levels of the targeted RNAs. In some embodiments,methods disclosed herein may also or alternatively involve increasingtranslation or increasing transcription of targeted RNAs, therebyelevating levels of RNA and/or protein levels in a targeted manner.

It is recognized that certain RNA degradation is mediated byexonucleases. In some embodiments, exonucleases may destroy RNA from its3′ end and/or 5′ end. Without wishing to be bound by theory, in someembodiments, it is believed that one or both ends of RNA can beprotected from exonuclease enzyme activity by contacting the RNA witholigonucleotides (oligos) that hybridize with the RNA at or near one orboth ends, thereby increasing stability and/or levels of the RNA. Theability to increase stability and/or levels of a RNA by targeting theRNA at or near one or both ends, as disclosed herein, is surprising inpart because of the presence of endonucleases (e.g., in cells) capableof destroying the RNA through internal cleavage. Moreover, in someembodiments, it is surprising that a 5′ targeting oligonucleotide iseffective alone (e.g., not in combination with a 3′ targetingoligonucleotide or in the context of a pseudocircularizationoligonucleotide) at stabilizing RNAs or increasing RNA levels because incells, for example, 3′ end processing exonucleases may be dominant(e.g., compared with 5′ end processing exonucleases). However, in someembodiments, 3′ targeting oligonucleotides are used in combination with5′ targeting oligonucleotides, or alone, to stabilize a target RNA.

In some embodiments, methods provided herein involve use ofoligonucleotides that stabilize an RNA by hybridizing at a 5′ and/or 3′region of the RNA. In some embodiments, oligonucleotides that prevent orinhibit degradation of an RNA by hybridizing with the RNA may bereferred to herein as “stabilizing oligonucleotides.” In some examples,such oligonucleotides hybridize with an RNA and prevent or inhibitexonuclease mediated degradation. Inhibition of exonuclease mediateddegradation includes, but is not limited to, reducing the extent ofdegradation of a particular RNA by exonucleases. For example, anexonuclease that processes only single stranded RNA may cleave a portionof the RNA up to a region where an oligonucleotide is hybridized withthe RNA because the exonuclease cannot effectively process (e.g., passthrough) the duplex region. Thus, in some embodiments, using anoligonucleotide that targets a particular region of an RNA makes itpossible to control the extent of degradation of the RNA by exonucleasesup to that region.

For example, use of an oligonucleotide (oligo) that hybridizes at an endof an RNA may reduce or eliminate degradation by an exonuclease thatprocesses only single stranded RNAs from that end. For example, use ofan oligonucleotide that hybridizes at the 5′ end of an RNA may reduce oreliminate degradation by an exonuclease that processes single strandedRNAs in a 5′ to 3′ direction. Similarly, use of an oligonucleotide thathybridizes at the 3′ end of an RNA may reduce or eliminate degradationby an exonuclease that processes single stranded RNAs in a 3′ to 5′direction. In some embodiments, lower concentrations of an oligo may beused when the oligo hybridizes at both the 5′ and 3′ regions of the RNA.In some embodiments, an oligo that hybridizes at both the 5′ and 3′regions of the RNA protects the 5′ and 3′ regions of the RNA fromdegradation (e.g., by an exonuclease). In some embodiments, an oligothat hybridizes at both the 5′ and 3′ regions of the RNA creates apseudo-circular RNA (e.g., a circularized RNA with a region of the polyAtail that protrudes from the circle). In some embodiments, apseudo-circular RNA is translated at a higher efficiency than anon-pseudo-circular RNA.

In some aspects, methods are provided for stabilizing a synthetic RNAdisclosed herein (e.g., a synthetic RNA that is to be delivered to acell). In some embodiments, the methods involve contacting a syntheticRNA with one or more oligonucleotides that bind to a 5′ region of thesynthetic RNA and a 3′ region of the synthetic RNA and that when boundto the synthetic RNA form a circularized product with the synthetic RNA.In some embodiments, the synthetic RNA is contacted with the one or moreoligonucleotides outside of a cell. In some embodiments, the methodsfurther involve delivering the circularized product to a cell.

In some aspects of the invention, methods are provided for increasingexpression of a protein in a cell that involve delivering to a cell acircularized synthetic RNA that encodes the protein, in which synthesisof the protein in the cell is increased following delivery of thecircularized RNA to the cell. In some embodiments, the circularizedsynthetic RNA comprises one or more modified nucleotides. In someembodiments, methods are provided that involve delivering to a cell acircularized synthetic RNA that encodes a protein, in which synthesis ofthe protein in the cell is increased following delivery of thecircularized synthetic RNA to the cell. In some embodiments, acircularized synthetic RNA is a single-stranded covalently closedcircular RNA. In some embodiments, a single-stranded covalently closedcircular RNA comprises one or more modified nucleotides. In someembodiments, the circularized synthetic RNA is formed by synthesizing anRNA that has a 5′ end and a 3′ and ligating together the 5′ and 3′ ends.In some embodiments, the circularized synthetic RNA is formed byproducing a synthetic RNA (e.g., through in vitro transcription orartificial (non-natural) chemical synthesis) and contacting thesynthetic RNA with one or more oligonucleotides that bind to a 5′ regionof the synthetic RNA and a 3′ region of the synthetic RNA, and that whenbound to the synthetic RNA form a circularized product with thesynthetic RNA.

In some aspects of the invention, an oligonucleotide is provided thatcomprises a region of complementarity that is complementary with atleast 5 contiguous nucleotides of an RNA transcript, in which thenucleotide at the 3′-end of the region of complementary is complementarywith a nucleotide within 10 nucleotides of the transcription start siteof the RNA transcript. In some embodiments, the oligonucleotidecomprises nucleotides linked by at least one modified internucleosidelinkage or at least one bridged nucleotide. In some embodiments, theoligonucleotide is 8 to 80, 8 to 50, 9 to 50, 10 to 50, 8 to 30, 9 to30, 10 to 30, 15 to 30, 9 to 20, 8 to 20, 8 to 15, or 9 to 15nucleotides in length. In some embodiments, the oligonucleotide is 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80 or more nucleotides inlength.

In some aspects of the invention, an oligonucleotide is provided thatcomprises two regions of complementarity each of which is complementarywith at least 5 contiguous nucleotides of an RNA transcript, in whichthe nucleotide at the 3′-end of the first region of complementary iscomplementary with a nucleotide within 100 nucleotides of thetranscription start site of the RNA transcript and in which the secondregion of complementarity is complementary with a region of the RNAtranscript that ends within 300 nucleotides of the 3′-end of the RNAtranscript.

Several exemplary oligonucleotide design schemes are contemplated hereinfor increasing stability of the RNA (e.g., mRNA) molecules disclosedherein. With regard to oligonucleotides targeting the 3′ end of an RNA,at least two exemplary design schemes are contemplated. As a firstscheme, an oligonucleotide is designed to be complementary to the 3′ endof an RNA, before the polyA tail. As a second scheme, an oligonucleotideis designed to be complementary to the 3′ end of RNA and theoligonucleotide has a 5′ poly-T region that hybridizes to the polyA tailof the RNA.

With regard to oligonucleotides targeting the 5′ end of an RNA, at leastthree exemplary design schemes are contemplated. For scheme one, anoligonucleotide is designed to be complementary to the 5′ end of RNA.For scheme two, an oligonucleotide is designed to be complementary tothe 5′ end of RNA and has a 3 ‘overhang to create a RNA-oligo duplexwith a recessed end. In this scheme, the overhang is one or more Cnucleotides, e.g., two Cs, which can potentially interact with a 5’methylguanosine cap and stabilize the cap further. The overhang couldalso potentially be another type of nucleotide, and is not limited to C.For scheme three, an oligonucleotide is designed to include a loopregion to stabilize a 5′ RNA cap. The example shows oligos with loops tostabilize a 5′ RNA cap or oligos. In yet another embodiment, anoligonucleotide is designed to bind to both 5′ and 3′ ends of an RNA tocreate a pseudo-circularized RNA. For example, an LNA mixmer oligobinding to the 5′ and 3′ regions of an RNA can achieve an oligo-mediatedRNA pseudo circularization.

An oligonucleotide designed as described above may be tested for itsability to upregulate RNA by increasing mRNA stability using the methodsoutlined in US20150050738A1 and WO2015023975A1, the contents of each ofwhich are herein incorporated by reference in their entireties.

Provided are methods of inducing translation of a syntheticpolynucleotide (e.g., a modified mRNA as disclosed herein) to produce apolypeptide in a cell population using the mRNAs described herein. Suchtranslation can be in vivo, ex vivo, in culture, or in vitro. The cellpopulation is contacted with an effective amount of a compositioncontaining a polynucleotide that incorporates the cap analog of thedisclosure, and a translatable region encoding the polypeptide. Thepopulation is contacted under conditions such that the polynucleotide islocalized into one or more cells of the cell population and thepolypeptide is translated in the cell from the polynucleotide.

An effective amount of the composition of a polynucleotide disclosedherein is provided based, at least in part, on the target tissue, targetcell type, means of administration, physical characteristics of thepolynucleotide (e.g., size, and extent of modified nucleosides), andother determinants. In general, an effective amount of the compositionprovides efficient protein production in the cell, preferably moreefficient than a composition containing a corresponding naturalpolynucleotide. Increased efficiency may be demonstrated by increasedcell transfection (i.e., the percentage of cells transfected with thepolynucleotide), increased protein translation from the polynucleotide,decreased polynucleotide degradation (as demonstrated, e.g., byincreased duration of protein translation from an RNA molecule), orreduced innate immune response of the host cell or improve therapeuticutility.

Aspects of the present disclosure are directed to methods of inducing invivo translation of a polypeptide in a mammalian subject in needthereof. Therein, an effective amount of a composition containing apolynucleotide of the disclosure that has the cap analog of thedisclosure and a translatable region encoding the polypeptide isadministered to the subject using the delivery methods described herein.The polynucleotide may also contain at least one modified nucleoside.The polynucleotide is provided in an amount and under other conditionssuch that the polynucleotide is localized into a cell or cells of thesubject and the polypeptide of interest is translated in the cell fromthe polynucleotide. The cell in which the polynucleotide is localized,or the tissue in which the cell is present, may be targeted with one ormore than one rounds of polynucleotide administration.

Other aspects of the present disclosure relate to transplantation ofcells containing RNA molecules of the disclosure to a mammalian subject.Administration of cells to mammalian subjects is known to those ofordinary skill in the art, such as local implantation (e.g., topical orsubcutaneous administration), organ delivery or systemic injection(e.g., intravenous injection or inhalation), as is the formulation ofcells in pharmaceutically acceptable carrier. Compositions containingRNA molecules of the disclosure are formulated for administrationintramuscularly, transarterially, intraperitoneally, intravenously,intranasally, subcutaneously, endoscopically, transdermally, orintrathecally. In some embodiments, the composition is formulated forextended release.

The subject to whom the therapeutic agent is administered suffers fromor is at risk of developing a disease, disorder, or deleteriouscondition. Provided are methods of identifying, diagnosing, andclassifying subjects on these bases, which may include clinicaldiagnosis, biomarker levels, genome-wide association studies (GWAS), andother methods known in the art.

In certain embodiments, the administered RNA molecule of the disclosuredirects production of one or more polypeptides that provide a functionalactivity which is substantially absent in the cell in which thepolypeptide is translated. For example, the missing functional activitymay be enzymatic, structural, or gene regulatory in nature.

In other embodiments, the administered RNA molecule of the disclosuredirects production of one or more polypeptides that replace apolypeptide (or multiple polypeptides) that is substantially absent inthe cell in which the one or more polypeptides are translated. Suchabsence may be due to genetic mutation of the encoding gene orregulatory pathway thereof. In other embodiments, the administered RNAmolecule of the disclosure directs production of one or morepolypeptides to supplement the amount of polypeptide (or multiplepolypeptides) that is present in the cell in which the one or morepolypeptides are translated. Alternatively, the translated polypeptidefunctions to antagonize the activity of an endogenous protein presentin, on the surface of, or secreted from the cell. Usually, the activityof the endogenous protein is deleterious to the subject, for example,due to mutation of the endogenous protein resulting in altered activityor localization. Additionally, the translated polypeptide antagonizes,directly or indirectly, the activity of a biological moiety present in,on the surface of, or secreted from the cell. Examples of antagonizedbiological moieties include lipids (e.g., cholesterol), a lipoprotein(e.g., low density lipoprotein), a polynucleotide, a carbohydrate, or asmall molecule toxin.

The translated proteins described herein are engineered for localizationwithin the cell, potentially within a specific compartment such as thenucleus, or are engineered for secretion from the cell or translocationto the plasma membrane of the cell.

As described herein, a useful feature of the RNA molecules of thedisclosure of the present disclosure is the capacity to reduce, evade,avoid or eliminate the innate immune response of a cell to an exogenousRNA. Provided are methods for performing the titration, reduction orelimination of the immune response in a cell or a population of cells.In some embodiments, the cell is contacted with a first composition thatcontains a first dose of a first exogenous RNA including a translatableregion, the cap analog of the disclosure, and optionally at least onemodified nucleoside, and the level of the innate immune response of thecell to the first exogenous polynucleotide is determined. Subsequently,the cell is contacted with a second composition, which includes a seconddose of the first exogenous polynucleotide, the second dose containing alesser amount of the first exogenous polynucleotide as compared to thefirst dose. Alternatively, the cell is contacted with a first dose of asecond exogenous polynucleotide. The second exogenous polynucleotide maycontain the cap analog of the disclosure, which may be the same ordifferent from the first exogenous polynucleotide or, alternatively, thesecond exogenous polynucleotide may not contain the cap analog of thedisclosure. The steps of contacting the cell with the first compositionand/or the second composition may be repeated one or more times.Additionally, efficiency of protein production (e.g., proteintranslation) in the cell is optionally determined, and the cell may bere-transfected with the first and/or second composition repeatedly untila target protein production efficiency is achieved.

Also provided herein are methods for treating or preventing a symptom ofdiseases characterized by missing or aberrant protein activity, byreplacing the missing protein activity or overcoming the aberrantprotein activity. Because of the rapid initiation of protein productionfollowing introduction of unnatural mRNAs, as compared to viral DNAvectors, the compounds and RNAs of the present disclosure areparticularly advantageous in treating acute diseases such as sepsis,stroke, and myocardial infarction. Moreover, the lack of transcriptionalregulation of the unnatural mRNAs of the present disclosure isadvantageous in that accurate titration of protein production isachievable. Multiple diseases are characterized by missing (orsubstantially diminished such that proper protein function does notoccur) protein activity. Such proteins may not be present, are presentin very low quantities or are essentially non-functional. The presentdisclosure provides a method for treating such conditions or diseases ina subject by introducing polynucleotide or cell-based therapeuticscontaining the RNA molecules of the disclosure provided herein, whereinthe RNA molecules of the disclosure encode for a protein that replacesthe protein activity missing from the target cells of the subject.

Diseases characterized by dysfunctional or aberrant protein activityinclude, but not limited to, cancer and proliferative diseases, geneticdiseases (e.g., cystic fibrosis), autoimmune diseases, diabetes,neurodegenerative diseases, cardiovascular diseases, and metabolicdiseases. The present disclosure provides a method for treating suchconditions or diseases in a subject by introducing the RNA molecules ofthe disclosure or cell-based therapeutics containing the RNA moleculesprovided herein, wherein the RNA molecules of the disclosure encode fora protein that antagonizes or otherwise overcomes the aberrant proteinactivity present in the cell of the subject.

Specific examples of a dysfunctional protein are the missense ornonsense mutation variants of the cystic fibrosis transmembraneconductance regulator (CFTR) gene, which produce a dysfunctional ornonfunctional, respectively, protein variant of CFTR protein, whichcauses cystic fibrosis.

Thus, provided are methods of treating cystic fibrosis in a mammaliansubject by contacting a cell of the subject with an RNA molecule of thedisclosure having a translatable region that encodes a functional CFTRpolypeptide, under conditions such that an effective amount of the CTFRpolypeptide is present in the cell. Preferred target cells areepithelial cells, such as the lung, and methods of administration aredetermined in view of the target tissue; i.e., for lung delivery, theRNA molecules are formulated for administration by inhalation.

In another embodiment, the present disclosure provides a method fortreating hyperlipidemia in a subject, by introducing into a cellpopulation of the subject with an unnatural mRNA molecule encodingSortilin, a protein recently characterized by genomic studies, therebyameliorating the hyperlipidemia in a subject. The SORT1 gene encodes atrans-Golgi network (TGN) transmembrane protein called Sortilin. Geneticstudies have shown that one of five individuals has a single nucleotidepolymorphism, rs12740374, in the 1p13 locus of the SORT1 gene thatpredisposes them to having low levels of low-density lipoprotein (LDL)and very-low-density lipoprotein (VLDL). Each copy of the minor allele,present in about 30% of people, alters LDL cholesterol by 8 mg/dL, whiletwo copies of the minor allele, present in about 5% of the population,lowers LDL cholesterol 16 mg/dL. Carriers of the minor allele have alsobeen shown to have a 40% decreased risk of myocardial infarction.Functional in vivo studies in mice describes that overexpression ofSORT1 in mouse liver tissue led to significantly lower LDL-cholesterollevels, as much as 80% lower, and that silencing SORT1 increased LDLcholesterol approximately 200% (Musunuru K et al. From noncoding variantto phenotype via SORT1 at the 1p13 cholesterol locus. Nature 2010; 466:714-721).

Methods of the present disclosure may enhance polynucleotide deliveryinto a cell population, in vivo, ex vivo, or in culture. For example, acell culture containing a plurality of host cells (e.g., eukaryoticcells such as yeast or mammalian cells) is contacted with a compositionthat contains an RNA molecule disclosed herein. The composition alsogenerally contains a transfection reagent or other compound thatincreases the efficiency of RNA uptake into the host cells. The RNAs ofthe disclosure may exhibit enhanced retention in the cell population,relative to a corresponding natural polynucleotide. For example, theretention of the RNA of the disclosure is greater than the retention ofthe corresponding polynucleotide. In some embodiments, it is at leastabout 50%, 75%, 90%, 95%, 100%, 150%, 200% or more than 200% greaterthan the retention of the natural polynucleotide. Such retentionadvantage may be achieved by one round of transfection with the RNA ofthe disclosure, or may be obtained following repeated rounds oftransfection.

In some embodiments, the RNA of the disclosure is delivered to a targetcell population with one or more additional polynucleotides. Suchdelivery may be at the same time, or the RNA of the disclosure isdelivered prior to delivery of the one or more additionalpolynucleotides. The additional one or more polynucleotides may be RNAmolecules of the disclosure or natural polynucleotides. It is understoodthat the initial presence of the RNA of the disclosure does notsubstantially induce an innate immune response of the cell populationand, moreover, that the innate immune response will not be activated bythe later presence of the natural polynucleotides. In this regard, theRNA of the disclosure may not itself contain a translatable region, ifthe protein desired to be present in the target cell population istranslated from the natural polynucleotides.

The present disclosure also provides proteins generated from unnaturalmRNAs.

The present disclosure provides pharmaceutical compositions of the RNAmolecules or multimeric structures disclosed herein, optionally incombination with one or more pharmaceutically acceptable excipients. Thepresent disclosure also provides pharmaceutical compositions of proteinsgenerated from the RNA molecules or multimeric structures disclosedherein, optionally in combination with one or more pharmaceuticallyacceptable excipients. Pharmaceutical compositions may optionallycomprise one or more additional active substances, e.g., therapeuticallyand/or prophylactically active substances. Pharmaceutical compositionsof the present disclosure may be sterile and/or pyrogen-free. Generalconsiderations in the formulation and/or manufacture of pharmaceuticalagents may be found, for example, in Remington: The Science and Practiceof Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporatedherein by reference in its entirety).

Pharmaceutical compositions may optionally comprise one or moreadditional therapeutically active substances. In accordance with someembodiments, a method of administering pharmaceutical compositionscomprising an RNA of the disclosure, encoding one or more proteins to bedelivered to a subject in need thereof is provided. In some embodiments,compositions are administered to humans. For the purposes of the presentdisclosure, the phrase “active ingredient” generally refers to apolynucleotide (e.g., an mRNA encoding polynucleotide to be delivered),a multimeric structure, a protein, protein encoding orprotein-containing complex as described herein and salts thereof.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts.

Modification of pharmaceutical compositions suitable for administrationto humans in order to render the compositions suitable foradministration to various animals is well understood, and the ordinarilyskilled veterinary pharmacologist can design and/or perform suchmodification with merely ordinary, if any, experimentation. Subjects towhich administration of the pharmaceutical compositions is contemplatedinclude, but are not limited to, humans and/or other primates; mammals,including commercially relevant mammals such as cattle, pigs, horses,sheep, cats, dogs, mice, and/or rats; and/or birds, includingcommercially relevant birds such as chickens, ducks, geese, and/orturkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, shaping and/or packaging the product into a desired single-or multi-dose unit.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” is discrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosurewill vary, depending upon the identity, size, and/or condition of thesubject treated and further depending upon the route by which thecomposition is to be administered. By way of example, the compositionmay comprise between 0.1% and 100% (w/w), e.g., between 0.1% and 99%,between 0.5 and 50%, between 1-30%, between 5-80%, or at least 80%(w/w), active ingredient.

The polynucleotides and multimeric structures of the disclosure can beformulated using one or more excipients to: (1) increase stability; (2)increase cell transfection; (3) permit the sustained or delayed release(e.g., from a depot formulation); (4) alter the biodistribution (e.g.,target to specific tissues or cell types); (5) increase the translationof encoded protein in vivo; and/or (6) alter the release profile ofencoded protein in vivo. In addition to traditional excipients such asany and all solvents, dispersion media, diluents, or other liquidvehicles, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, excipients ofthe present disclosure can include, without limitation, lipidoids,liposomes, lipid nanoparticles, polymers, lipoplexes, core-shellnanoparticles, peptides, proteins, cells transfected with multimericstructures, hyaluronidase, nanoparticle mimics and combinations thereof.

In some embodiments, the nucleic acids (e.g., mRNAs, or IVT mRNAs) andmultimeric nucleic acid molecules of the disclosure (e.g., multimericmRNA molecules) can be formulated using one or more liposomes,lipoplexes, or lipid nanoparticles. In one embodiment, pharmaceuticalcompositions of the nucleic acids or multimeric nucleic acid moleculesinclude lipid nanoparticles (LNPs). In some embodiments, lipidnanoparticles are MC3-based lipid nanoparticles.

The number of polynucleotides encapsulated by a lipid nanoparticleranges from about 1 polynucleotide to about 100 polynucleotides. In someembodiments, the number of polynucleotides encapsulated by a lipidnanoparticle ranges from about 50 to about 500 polynucleotides. In someembodiments, the number of polynucleotides encapsulated by a lipidnanoparticle ranges from about 250 to about 1000 polynucleotides. Insome embodiments, the number of polynucleotides encapsulated by a lipidnanoparticle is greater than 1000.

The number of multimeric molecules encapsulated by a lipid nanoparticleranges from about 1 multimeric molecule to about 100 multimericmolecules. In some embodiments, the number of multimeric moleculesencapsulated by a lipid nanoparticle ranges from about 50 multimericmolecules to about 500 multimeric molecules. In some embodiments, thenumber of multimeric molecules encapsulated by a lipid nanoparticleranges from about 250 multimeric molecules to about 1000 multimericmolecules. In some embodiments, the number of multimeric moleculesencapsulated by a lipid nanoparticle is greater than 1000 multimericmolecules.

In one embodiment, the polynucleotides or multimeric structures may beformulated in a lipid-polycation complex. The formation of thelipid-polycation complex may be accomplished by methods known in theart. As a non-limiting example, the polycation may include a cationicpeptide or a polypeptide such as, but not limited to, polylysine,polyornithine and/or polyarginine. In another embodiment, thepolynucleotides or multimeric structures may be formulated in alipid-polycation complex which may further include a non-cationic lipidsuch as, but not limited to, cholesterol ordioleoylphosphatidylethanolamine (DOPE).

The liposome formulation may be influenced by, but not limited to, theselection of the cationic lipid component, the degree of cationic lipidsaturation, the nature of the PEGylation, ratio of all components andbiophysical parameters such as size. In one example by Semple et al.(Semple et al. Nature Biotech. 2010 28:172-176; herein incorporated byreference in its entirety), the liposome formulation is composed of57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3%cholesterol, and 1.4% PEG-c-DMA. As another example, changing thecomposition of the cationic lipid could more effectively deliver siRNAto various antigen presenting cells (Basha et al. Mol Ther. 201119:2186-2200; herein incorporated by reference in its entirety). In someembodiments, liposome formulations may comprise from about 35 to about45% cationic lipid, from about 40% to about 50% cationic lipid, fromabout 50% to about 60% cationic lipid and/or from about 55% to about 65%cationic lipid. In some embodiments, the ratio of lipid to mRNA inliposomes may be from about 5:1 to about 20:1, from about 10:1 to about25:1, from about 15:1 to about 30:1 and/or at least 30:1.

In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP)formulations may be increased or decreased and/or the carbon chainlength of the PEG lipid may be modified from C14 to C18 to alter thepharmacokinetics and/or biodistribution of the LNP formulations. As anon-limiting example, LNP formulations may contain from about 0.5% toabout 3.0%, from about 1.0% to about 3.5%, from about 1.5% to about4.0%, from about 2.0% to about 4.5%, from about 2.5% to about 5.0%and/or from about 3.0% to about 6.0% of the lipid molar ratio ofPEG-c-DOMG(R-3-[(ω)-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine)(also referred to herein as PEG-DOMG) as compared to the cationic lipid,DSPC and cholesterol. In another embodiment the PEG-c-DOMG may bereplaced with a PEG lipid such as, but not limited to, PEG-DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG(1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid may be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In one embodiment, the polynucleotides or multimeric structuresdisclosed herein are formulated in a nanoparticle which may comprise atleast one lipid. The lipid may be selected from, but is not limited to,DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA,DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. Inanother aspect, the lipid may be a cationic lipid such as, but notlimited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA andamino alcohol lipids. The amino alcohol cationic lipid may be the lipidsdescribed in and/or made by the methods described in US PatentPublication No. US20130150625, herein incorporated by reference in itsentirety. As a non-limiting example, the cationic lipid may be2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methylpropan-1-ol (Compound 1 in US20130150625);2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol(Compound 2 in US20130150625);2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol(Compound 3 in US20130150625); and2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 4 in US20130150625); or any pharmaceutically acceptable saltor stereoisomer thereof.

Lipid nanoparticle formulations typically comprise a lipid, inparticular, an ionizable cationic lipid, for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), ordi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and furthercomprise a neutral lipid, a sterol and a molecule capable of reducingparticle aggregation, for example a PEG or PEG-modified lipid.

In one embodiment, the lipid nanoparticle formulation consistsessentially of (i) at least one lipid selected from the group consistingof 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) aneutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) asterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG orPEG-cDMA, in a molar ratio of about 20-60% cationic lipid: 5-25% neutrallipid: 25-55% sterol; 0.5-15% PEG-lipid.

In one embodiment, the formulation includes from about 25% to about 75%on a molar basis of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., fromabout 35 to about 65%, from about 45 to about 65%, about 60%, about57.5%, about 50% or about 40% on a molar basis.

In one embodiment, the formulation includes from about 0.5% to about 15%on a molar basis of the neutral lipid e.g., from about 3 to about 12%,from about 5 to about 10% or about 15%, about 10%, or about 7.5% on amolar basis. Exemplary neutral lipids include, but are not limited to,DSPC, POPC, DPPC, DOPE and SM. In one embodiment, the formulationincludes from about 5% to about 50% on a molar basis of the sterol(e.g., about 15 to about 45%, about 20 to about 40%, about 40%, about38.5%, about 35%, or about 31% on a molar basis. An exemplary sterol ischolesterol. In one embodiment, the formulation includes from about 0.5%to about 20% on a molar basis of the PEG or PEG-modified lipid (e.g.,about 0.5 to about 10%, about 0.5 to about 5%, about 1.5%, about 0.5%,about 1.5%, about 3.5%, or about 5% on a molar basis. In one embodiment,the PEG or PEG modified lipid comprises a PEG molecule of an averagemolecular weight of 2,000 Da. In other embodiments, the PEG or PEGmodified lipid comprises a PEG molecule of an average molecular weightof less than 2,000 Da, for example around 1,500 Da, around 1,000 Da, oraround 500 Da. Exemplary PEG-modified lipids include, but are notlimited to, PEG-distearoyl glycerol (PEG-DMG) (also referred herein asPEG-C14 or C14-PEG), PEG-cDMA.

In one embodiment, the formulations disclosed herein include 25-75% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% ofthe neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations disclosed herein include 35-65% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of theneutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations disclosed herein include 45-65% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of theneutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations disclosed herein include about 60%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5%of the neutral lipid, about 31% of the sterol, and about 1.5% of the PEGor PEG-modified lipid on a molar basis.

In one embodiment, the formulations disclosed herein include about 50%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofthe neutral lipid, about 38.5% of the sterol, and about 1.5% of the PEGor PEG-modified lipid on a molar basis.

In one embodiment, the formulations disclosed herein include about 50%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofthe neutral lipid, about 35% of the sterol, about 4.5% or about 5% ofthe PEG or PEG-modified lipid, and about 0.5% of the targeting lipid ona molar basis.

In one embodiment, the formulations disclosed herein include about 40%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 15% ofthe neutral lipid, about 40% of the sterol, and about 5% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations disclosed herein include about 57.2%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.1%of the neutral lipid, about 34.3% of the sterol, and about 1.4% of thePEG or PEG-modified lipid on a molar basis.

In one embodiment, the formulations disclosed herein include about 57.5%of a cationic lipid selected from the PEG lipid is PEG-cDMA (PEG-cDMA isfurther discussed in Reyes et al. (J. Controlled Release, 107, 276-287(2005), the contents of which are herein incorporated by reference inits entirety), about 7.5% of the neutral lipid, about 31.5% of thesterol, and about 3.5% of the PEG or PEG-modified lipid on a molarbasis.

In preferred embodiments, lipid nanoparticle formulation consistsessentially of a lipid mixture in molar ratios of about 20-70% cationiclipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modifiedlipid; more preferably in a molar ratio of about 20-60% cationic lipid:5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid.

In particular embodiments, the molar lipid ratio is approximately50/10/38.5/1.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG-DPG),57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid, e.g.,DPPC/Chol/PEG-modified lipid, e.g., PEG-cDMA), 40/15/40/5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DSG), 50/10/35/5 (cationiclipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG),40/10/40/10 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35/15/40/10(mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid,e.g., PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol % cationic lipid/neutrallipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

Exemplary lipid nanoparticle compositions and methods of making same aredescribed, for example, in Semple et al. (2010) Nat. Biotechnol.28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (thecontents of each of which are incorporated herein by reference in theirentirety).

In one embodiment, the lipid nanoparticle formulations described hereinmay comprise a cationic lipid, a PEG lipid and a structural lipid andoptionally comprise a non-cationic lipid. As a non-limiting example, thelipid nanoparticle may comprise about 40-60% of cationic lipid, about5-15% of a non-cationic lipid, about 1-2% of a PEG lipid and about30-50% of a structural lipid. As another non-limiting example, the lipidnanoparticle may comprise about 50% cationic lipid, about 10%non-cationic lipid, about 1.5% PEG lipid and about 38.5% structurallipid. As yet another non-limiting example, the lipid nanoparticle maycomprise about 55% cationic lipid, about 10% non-cationic lipid, about2.5% PEG lipid and about 32.5% structural lipid. In one embodiment, thecationic lipid may be any cationic lipid described herein such as, butnot limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.

In one embodiment, the lipid nanoparticle formulations described hereinmay be 4 component lipid nanoparticles. The lipid nanoparticle maycomprise a cationic lipid, a non-cationic lipid, a PEG lipid and astructural lipid. As a non-limiting example, the lipid nanoparticle maycomprise about 40-60% of cationic lipid, about 5-15% of a non-cationiclipid, about 1-2% of a PEG lipid and about 30-50% of a structural lipid.As another non-limiting example, the lipid nanoparticle may compriseabout 50% cationic lipid, about 10% non-cationic lipid, about 1.5% PEGlipid and about 38.5% structural lipid. As yet another non-limitingexample, the lipid nanoparticle may comprise about 55% cationic lipid,about 10% non-cationic lipid, about 2.5% PEG lipid and about 32.5%structural lipid. In one embodiment, the cationic lipid may be anycationic lipid described herein such as, but not limited to,DLin-KC2-DMA, DLin-MC3-DMA and L319.

In one embodiment, the lipid nanoparticle formulations described hereinmay comprise a cationic lipid, a non-cationic lipid, a PEG lipid and astructural lipid. As a non-limiting example, the lipid nanoparticlecomprise about 50% of the cationic lipid DLin-KC2-DMA, about 10% of thenon-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DOMG and about38.5% of the structural lipid cholesterol. As a non-limiting example,the lipid nanoparticle comprise about 50% of the cationic lipidDLin-MC3-DMA, about 10% of the non-cationic lipid DSPC, about 1.5% ofthe PEG lipid PEG-DOMG and about 38.5% of the structural lipidcholesterol. As a non-limiting example, the lipid nanoparticle compriseabout 50% of the cationic lipid DLin-MC3-DMA, about 10% of thenon-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DMG and about38.5% of the structural lipid cholesterol. As yet another non-limitingexample, the lipid nanoparticle comprise about 55% of the cationic lipidL319, about 10% of the non-cationic lipid DSPC, about 2.5% of the PEGlipid PEG-DMG and about 32.5% of the structural lipid cholesterol.

In one embodiment, the polynucleotides or multimeric molecules (e.g.,multimeric mRNA molecules) of the disclosure may be formulated in lipidnanoparticles having a diameter from about 10 to about 100 nm such as,but not limited to, about 10 to about 20 nm, about 10 to about 30 nm,about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 toabout 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm,about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 toabout 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50to about 70 nm about 50 to about 80 nm, about 50 to about 90 nm, about50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm,about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about90 nm, about 80 to about 100 nm and/or about 90 to about 100 nm.

In one embodiment, the lipid nanoparticles may have a diameter fromabout 10 to 500 nm. In one embodiment, the lipid nanoparticle may have adiameter greater than 100 nm, greater than 150 nm, greater than 200 nm,greater than 250 nm, greater than 300 nm, greater than 350 nm, greaterthan 400 nm, greater than 450 nm, greater than 500 nm, greater than 550nm, greater than 600 nm, greater than 650 nm, greater than 700 nm,greater than 750 nm, greater than 800 nm, greater than 850 nm, greaterthan 900 nm, greater than 950 nm or greater than 1000 nm. In someembodiments, the cationic lipid nanoparticle has a mean diameter of50-150 nm. In some embodiments, the cationic lipid nanoparticle has amean diameter of 80-100 nm.

In one embodiment, the compositions may comprise the polynucleotides ormultimeric polynucleotides described herein, formulated in a lipidnanoparticle comprising MC3, Cholesterol, DSPC and PEG2000-DMG, thebuffer trisodium citrate, sucrose and water for injection. As anon-limiting example, the composition comprises: 2.0 mg/mL of drugsubstance (e.g., multimeric polynucleotides), 21.8 mg/mL of MC3, 10.1mg/mL of cholesterol, 5.4 mg/mL of DSPC, 2.7 mg/mL of PEG2000-DMG, 5.16mg/mL of trisodium citrate, 71 mg/mL of sucrose and about 1.0 mL ofwater for injection.

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders, andlubricants, as suited to the particular dosage form desired. Remington'sThe Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro(Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporatedherein by reference) discloses various excipients used in formulatingpharmaceutical compositions and known techniques for the preparationthereof. Except insofar as any conventional excipient medium isincompatible with a substance or its derivatives, such as by producingany undesirable biological effect or otherwise interacting in adeleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thispresent disclosure.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humansand for veterinary use. In some embodiments, an excipient is approved byUnited States Food and Drug Administration. In some embodiments, anexcipient is pharmaceutical grade. In some embodiments, an excipientmeets the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical formulations.Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Other Components

A nanoparticle composition may include one or more components inaddition to those described in the preceding sections. For example, ananoparticle composition may include one or more small hydrophobicmolecules such as a vitamin (e.g., vitamin A or vitamin E) or a sterol.

Nanoparticle compositions may also include one or more permeabilityenhancer molecules, carbohydrates, polymers, surface altering agents, orother components. A permeability enhancer molecule may be a moleculedescribed by U.S. patent application publication No. 2005/0222064, forexample. Carbohydrates may include simple sugars (e.g., glucose) andpolysaccharides (e.g., glycogen and derivatives and analogs thereof).

A polymer may be included in and/or used to encapsulate or partiallyencapsulate a nanoparticle composition. A polymer may be biodegradableand/or biocompatible. A polymer may be selected from, but is not limitedto, polyamines, polyethers, polyamides, polyesters, polycarbamates,polyureas, polycarbonates, polystyrenes, polyimides, polysulfones,polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines,polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles,and polyarylates. For example, a polymer may include poly(caprolactone)(PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA),poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lacticacid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid)(PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA),poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP),polysiloxanes, polystyrene (PS), polyurethanes, derivatized cellulosessuch as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers,cellulose esters, nitro celluloses, hydroxypropylcellulose,carboxymethylcellulose, polymers of acrylic acids, such aspoly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate),poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate),poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate),poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methylacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate),poly(octadecyl acrylate) and copolymers and mixtures thereof,polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylenefumarate, polyoxymethylene, poloxamers, polyoxamines, poly(ortho)esters,poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone),trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM),poly(-methyl-2-oxazoline) (PMOX), poly(-ethyl-2-oxazoline) (PEOZ), andpolyglycerol.

Surface altering agents may include, but are not limited to, anionicproteins (e.g., bovine serum albumin), surfactants (e.g., cationicsurfactants such as dimethyldioctadecyl-ammonium bromide), sugars orsugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g.,heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g.,acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine,carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol,letosteine, stepronin, tiopronin, gelsolin, thymosin 134, dornase alfa,neltenexine, and erdosteine), and DNases (e.g., rhDNase). A surfacealtering agent may be disposed within a nanoparticle and/or on thesurface of a nanoparticle composition (e.g., by coating, adsorption,covalent linkage, or other process).

A nanoparticle composition may also comprise one or more functionalizedlipids. For example, a lipid may be functionalized with an alkyne groupthat, when exposed to an azide under appropriate reaction conditions,may undergo a cycloaddition reaction. In particular, a lipid bilayer maybe functionalized in this fashion with one or more groups useful infacilitating membrane permeation, cellular recognition, or imaging. Thesurface of a nanoparticle composition may also be conjugated with one ormore useful antibodies. Functional groups and conjugates useful intargeted cell delivery, imaging, and membrane permeation are well knownin the art.

In addition to these components, nanoparticle compositions of thedisclosure may include any substance useful in pharmaceuticalcompositions. For example, the nanoparticle composition may include oneor more pharmaceutically acceptable excipients or accessory ingredientssuch as, but not limited to, one or more solvents, dispersion media,diluents, dispersion aids, suspension aids, granulating aids,disintegrants, fillers, glidants, liquid vehicles, binders, surfaceactive agents, isotonic agents, thickening or emulsifying agents,buffering agents, lubricating agents, oils, preservatives, and otherspecies. Excipients such as waxes, butters, coloring agents, coatingagents, flavorings, and perfuming agents may also be included.Pharmaceutically acceptable excipients are well known in the art (seefor example Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md.,2006).

Examples of diluents may include, but are not limited to, calciumcarbonate, sodium carbonate, calcium phosphate, dicalcium phosphate,calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose,sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol,sorbitol, inositol, sodium chloride, dry starch, cornstarch, powderedsugar, and/or combinations thereof. Granulating and dispersing agentsmay be selected from the non-limiting list consisting of potato starch,corn starch, tapioca starch, sodium starch glycolate, clays, alginicacid, guar gum, citrus pulp, agar, bentonite, cellulose and woodproducts, natural sponge, cation-exchange resins, calcium carbonate,silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)(crospovidone), sodium carboxymethyl starch (sodium starch glycolate),carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate,quaternary ammonium compounds, and/or combinations thereof.

Surface active agents and/or emulsifiers may include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEEN® 60],polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate[SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate[SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ® 45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ® 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLURONIC® F 68, POLOXAMER® 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, and/or combinations thereof.

A binding agent may be starch (e.g. cornstarch and starch paste);gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses,lactose, lactitol, mannitol); natural and synthetic gums (e.g. acacia,sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilageof isapol husks, carboxymethylcellulose, methylcellulose,ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, microcrystalline cellulose, celluloseacetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®),and larch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; and combinations thereof, or any other suitable bindingagent.

Examples of preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Examples of antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Examples ofchelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Examples of antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Examples of antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Examples of alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, benzyl alcohol, phenol, phenoliccompounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethylalcohol. Examples of acidic preservatives include, but are not limitedto, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, aceticacid, dehydroascorbic acid, ascorbic acid, sorbic acid, and/or phyticacid. Other preservatives include, but are not limited to, tocopherol,tocopherol acetate, deteroxime mesylate, cetrimide, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine,sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodiumbisulfite, sodium metabisulfite, potassium sulfite, potassiummetabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115,GERMABEN® II, NEOLONE™, KATHON™, and/or EUXYL®.

Examples of buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconicacid, calcium glycerophosphate, calcium lactate, calcium lactobionate,propanoic acid, calcium levulinate, pentanoic acid, dibasic calciumphosphate, phosphoric acid, tribasic calcium phosphate, calciumhydroxide phosphate, potassium acetate, potassium chloride, potassiumgluconate, potassium mixtures, dibasic potassium phosphate, monobasicpotassium phosphate, potassium phosphate mixtures, sodium acetate,sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate,dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphatemixtures, tromethamine, amino-sulfonate buffers (e.g. HEPES), magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, and/or combinationsthereof. Lubricating agents may selected from the non-limiting groupconsisting of magnesium stearate, calcium stearate, stearic acid,silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils,polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride,leucine, magnesium lauryl sulfate, sodium lauryl sulfate, andcombinations thereof.

Examples of oils include, but are not limited to, almond, apricotkernel, avocado, babassu, bergamot, black current seed, borage, cade,camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter,coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, eveningprimrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut,hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender,lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoamseed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel,peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran,rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn,sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle,tsubaki, vetiver, walnut, and wheat germ oils as well as butyl stearate,caprylic triglyceride, capric triglyceride, cyclomethicone, diethylsebacate, dimethicone 360, simethicone, isopropyl myristate, mineraloil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinationsthereof.

Additional and Alternative Examples of Formulations

Nanoparticle compositions may include a lipid component and one or moreadditional components, such as a therapeutic agent. A nanoparticlecomposition may be designed for one or more specific applications ortargets. The elements of a nanoparticle composition may be selectedbased on a particular application or target, and/or based on theefficacy, toxicity, expense, ease of use, availability, or other featureof one or more elements. Similarly, the particular formulation of ananoparticle composition may be selected for a particular application ortarget according to, for example, the efficacy and toxicity ofparticular combinations of elements.

The lipid component of a nanoparticle composition of the disclosure mayinclude, for example, a lipid according to formula (I), a phospholipid(such as an unsaturated lipid, e.g., DOPE or DSPC), a PEG lipid, and astructural lipid. The elements of the lipid component may be provided inspecific fractions.

In some embodiments, the lipid component of a nanoparticle compositionincludes a lipid according to formula (I), a phospholipid, a PEG lipid,and a structural lipid. In certain embodiments, the lipid component ofthe nanoparticle composition includes about 30 mol % to about 60 mol %compound of formula (I), about 0 mol % to about 30 mol % phospholipid,about 18.5 mol % to about 48.5 mol % structural lipid, and about 0 mol %to about 10 mol % of PEG lipid, provided that the total mol % does notexceed 100%. In some embodiments, the lipid component of thenanoparticle composition includes about 35 mol % to about 55 mol %compound of formula (I), about 5 mol % to about 25 mol % phospholipid,about 30 mol % to about 40 mol % structural lipid, and about 0 mol % toabout 10 mol % of PEG lipid. In a particular embodiment, the lipidcomponent includes about 50 mol % said compound, about 10 mol %phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % ofPEG lipid. In another particular embodiment, the lipid componentincludes about 40 mol % said compound, about 20 mol % phospholipid,about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. Insome embodiments, the phospholipid may be DOPE or DSPC. In otherembodiments, the PEG lipid may be PEG-DMG and/or the structural lipidmay be cholesterol.

Nanoparticle compositions may be designed for one or more specificapplications or targets. For example, a nanoparticle composition may bedesigned to deliver a therapeutic agent such as an RNA to a particularcell, tissue, organ, or system or group thereof in a mammal's body.Physiochemical properties of nanoparticle compositions may be altered inorder to increase selectivity for particular bodily targets. Forinstance, particle sizes may be adjusted based on the fenestration sizesof different organs. The therapeutic agent included in a nanoparticlecomposition may also be selected based on the desired delivery target ortargets. For example, a therapeutic agent may be selected for aparticular indication, condition, disease, or disorder and/or fordelivery to a particular cell, tissue, organ, or system or group thereof(e.g., localized or specific delivery). In certain embodiments, ananoparticle composition may include an mRNA encoding a polypeptide ofinterest capable of being translated within a cell to produce thepolypeptide of interest. Such a composition may be designed to bespecifically delivered to a particular organ. In particular embodiments,a composition may be designed to be specifically delivered to amammalian liver.

The amount of a therapeutic agent in a nanoparticle composition maydepend on the size, composition, desired target and/or application, orother properties of the nanoparticle composition as well as on theproperties of the therapeutic agent. For example, the amount of an RNAuseful in a nanoparticle composition may depend on the size, sequence,and other characteristics of the RNA. The relative amounts of atherapeutic agent and other elements (e.g., lipids) in a nanoparticlecomposition may also vary. In some embodiments, the wt/wt ratio of thelipid component to a therapeutic agent in a nanoparticle composition maybe from about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1,35:1, 40:1, 45:1, 50:1, and 60:1. For example, the wt/wt ratio of thelipid component to a therapeutic agent may be from about 10:1 to about40:1. In preferred embodiments, the wt/wt ratio is about 20:1. Theamount of a therapeutic agent in a nanoparticle composition may, forexample, be measured using absorption spectroscopy (e.g.,ultraviolet-visible spectroscopy).

In some embodiments, a nanoparticle composition includes one or moreRNAs, and the one or more RNAs, lipids, and amounts thereof may beselected to provide a specific N:P ratio. The N:P ratio of thecomposition refers to the molar ratio of nitrogen atoms in one or morelipids to the number of phosphate groups in an RNA. In general, a lowerN:P ratio is preferred. The one or more RNA, lipids, and amounts thereofmay be selected to provide an N:P ratio from about 2:1 to about 30:1,such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1,18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, theN:P ratio may be from about 2:1 to about 8:1. In other embodiments, theN:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio maybe about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, orabout 7.0:1. For example, the N:P ratio may be about 5.67:1.

Physical Properties

The characteristics of a nanoparticle composition may depend on thecomponents thereof. For example, a nanoparticle composition includingcholesterol as a structural lipid may have different characteristicsthan a nanoparticle composition that includes a different structurallipid. Similarly, the characteristics of a nanoparticle composition maydepend on the absolute or relative amounts of its components. Forinstance, a nanoparticle composition including a higher molar fractionof a phospholipid may have different characteristics than a nanoparticlecomposition including a lower molar fraction of a phospholipid.Characteristics may also vary depending on the method and conditions ofpreparation of the nanoparticle composition.

Nanoparticle compositions may be characterized by a variety of methods.For example, microscopy (e.g., transmission electron microscopy orscanning electron microscopy) may be used to examine the morphology andsize distribution of a nanoparticle composition. Dynamic lightscattering or potentiometry (e.g., potentiometric titrations) may beused to measure zeta potentials. Dynamic light scattering may also beutilized to determine particle sizes. Instruments such as the ZetasizerNano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may alsobe used to measure multiple characteristics of a nanoparticlecomposition, such as particle size, polydispersity index, and zetapotential.

The mean size of a nanoparticle composition of the disclosure may bebetween 10s of nm and 100s of nm. For example, the mean size may be fromabout 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm,60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm,110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150nm. In some embodiments, the mean size of a nanoparticle composition maybe from about 50 nm to about 100 nm, from about 50 nm to about 90 nm,from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, fromabout 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nmto about 70 nm, from about 70 nm to about 100 nm, from about 70 nm toabout 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about100 nm. In certain embodiments, the mean size of a nanoparticlecomposition may be from about 70 nm to about 100 nm. In a particularembodiment, the mean size may be about 80 nm. In other embodiments, themean size may be about 100 nm.

A nanoparticle composition of the disclosure may be relativelyhomogenous. A polydispersity index may be used to indicate thehomogeneity of a nanoparticle composition, e.g., the particle sizedistribution of the nanoparticle compositions. A small (e.g., less than0.3) polydispersity index generally indicates a narrow particle sizedistribution. A nanoparticle composition of the disclosure may have apolydispersity index from about 0 to about 0.25, such as 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14,0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. Insome embodiments, the polydispersity index of a nanoparticle compositionmay be from about 0.10 to about 0.20.

The zeta potential of a nanoparticle composition may be used to indicatethe electrokinetic potential of the composition. For example, the zetapotential may describe the surface charge of a nanoparticle composition.Nanoparticle compositions with relatively low charges, positive ornegative, are generally desirable, as more highly charged species mayinteract undesirably with cells, tissues, and other elements in thebody. In some embodiments, the zeta potential of a nanoparticlecomposition of the disclosure may be from about −10 mV to about +20 mV,from about −10 mV to about +15 mV, from about −10 mV to about +10 mV,from about −10 mV to about +5 mV, from about −10 mV to about 0 mV, fromabout −10 mV to about −5 mV, from about −5 mV to about +20 mV, fromabout −5 mV to about +15 mV, from about −5 mV to about +10 mV, fromabout −5 mV to about +5 mV, from about −5 mV to about 0 mV, from about 0mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV toabout +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about+20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about+10 mV.

The efficiency of encapsulation of a therapeutic agent describes theamount of therapeutic agent that is encapsulated or otherwise associatedwith a nanoparticle composition after preparation, relative to theinitial amount provided. The encapsulation efficiency is desirably high(e.g., close to 100%). The encapsulation efficiency may be measured, forexample, by comparing the amount of therapeutic agent in a solutioncontaining the nanoparticle composition before and after breaking up thenanoparticle composition with one or more organic solvents ordetergents. Fluorescence may be used to measure the amount of freetherapeutic agent (e.g., RNA) in a solution. For the nanoparticlecompositions of the disclosure, the encapsulation efficiency of atherapeutic agent may be at least 50%, for example 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%. In some embodiments, the encapsulation efficiency may be at least80%. In certain embodiments, the encapsulation efficiency may be atleast 90%.

A nanoparticle composition disclosed herein may optionally comprise oneor more coatings. For example, a nanoparticle composition may beformulated in a capsule, film, or tablet having a coating. A capsule,film, or tablet including a composition of the disclosure may have anyuseful size, tensile strength, hardness, or density.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of an active ingredient of thepresent disclosure to alleviate the symptoms or complications of adisease, condition or disorder, or to eliminate the disease, conditionor disorder. The term “treat” can also include treatment of a cell invitro or an animal model.

An active ingredient of the present disclosure, can or may also be usedto prevent a relevant disease, condition or disorder, or used toidentify suitable candidates for such purposes. As used herein,“preventing,” “prevent,” or “protecting against” describes reducing oreliminating the onset of the symptoms or complications of such disease,condition or disorder.

As used herein, “combination therapy” or “co-therapy” includes theadministration of an active ingredient of the present disclosure, and atleast a second agent as part of a specific treatment regimen intended toprovide the beneficial effect from the co-action of these therapeuticagents. The beneficial effect of the combination includes, but is notlimited to, pharmacokinetic or pharmacodynamic co-action resulting fromthe combination of therapeutic agents.

A “pharmaceutical composition” is a formulation containing the activeingredient of the present disclosure in a form suitable foradministration to a subject. In one embodiment, the pharmaceuticalcomposition is in bulk or in unit dosage form. The unit dosage form isany of a variety of forms, including, for example, a capsule, an IV bag,a tablet, a single pump on an aerosol inhaler or a vial. The quantity ofactive ingredient (e.g., a formulation of the disclosed compound orsalt, hydrate, solvate or isomer thereof) in a unit dose of compositionis an effective amount and is varied according to the particulartreatment involved. One skilled in the art will appreciate that it issometimes necessary to make routine variations to the dosage dependingon the age and condition of the patient. The dosage will also depend onthe route of administration. A variety of routes are contemplated,including oral, pulmonary, rectal, parenteral, transdermal,subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational,buccal, sublingual, intrapleural, intrathecal, intranasal, and the like.Dosage forms for the topical or transdermal administration of an activeingredient of the disclosure include powders, sprays, ointments, pastes,creams, lotions, gels, solutions, patches and inhalants. In oneembodiment, the active compound is mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, anions, cations, materials, compositions, carriers, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

An active ingredient of the present disclosure can be administered to asubject in many of the well-known methods currently used forchemotherapeutic treatment. For example, for treatment of cancers, anactive ingredient of the present disclosure may be injected directlyinto tumors, injected into the blood stream or body cavities or takenorally or applied through the skin with patches. The dose chosen shouldbe sufficient to constitute effective treatment but not so high as tocause unacceptable side effects. The state of the disease condition(e.g., cancer, precancer, and the like) and the health of the patientshould preferably be closely monitored during and for a reasonableperiod after treatment.

An “effective amount” of the polynucleotides (e.g., RNA or mRNA) ormultimeric structures disclosed herein is based, at least in part, onthe target tissue, target cell type, means of administration, physicalcharacteristics of the polynucleotide (e.g., size, and extent ofmodified nucleosides) and other components of the multimeric structures,and other determinants. In general, an effective amount of RNA or themultimeric structure provides an induced or boosted peptide productionin the cell, preferably more efficient than a composition containing acorresponding unmodified polynucleotide encoding the same peptide orabout the same or more efficient than separate mRNAs that are not partof a multimeric structure. Increased peptide production may bedemonstrated by increased cell transfection (i.e., the percentage ofcells transfected with the multimeric structures), increased proteintranslation from the polynucleotide, decreased nucleic acid degradation(as demonstrated, e.g., by increased duration of protein translationfrom a modified polynucleotide), or altered peptide production in thehost cell.

The mRNA of the present disclosure may be designed to encodepolypeptides of interest selected from any of several target categoriesincluding, but not limited to, biologics, antibodies, vaccines,therapeutic proteins or peptides, cell penetrating peptides, secretedproteins, plasma membrane proteins, cytoplasmic or cytoskeletalproteins, intracellular membrane bound proteins, nuclear proteins,proteins associated with human disease, targeting moieties or thoseproteins encoded by the human genome for which no therapeutic indicationhas been identified but which nonetheless have utility in areas ofresearch and discovery. “Therapeutic protein” refers to a protein that,when administered to a cell has a therapeutic, diagnostic, and/orprophylactic effect and/or elicits a desired biological and/orpharmacological effect.

The term “therapeutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Therapeutically effective amounts for agiven situation can be determined by routine experimentation that iswithin the skill and judgment of the clinician. In a preferred aspect,the disease or condition to be treated is cancer. In another aspect, thedisease or condition to be treated is a cell proliferative disorder.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

In certain embodiments, compositions in accordance with the presentdisclosure may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg toabout 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic, diagnostic,prophylactic, or imaging. The desired dosage may be delivered threetimes a day, two times a day, once a day, every other day, every thirdday, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations). When multiple administrations are employed, splitdosing regimens such as those described herein may be used.

The pharmaceutical compositions containing active ingredient of thepresent disclosure may be manufactured in a manner that is generallyknown, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol and sorbitol, and sodium chloridein the composition. Prolonged absorption of the injectable compositionscan be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the active ingredient of the presentdisclosure is delivered in the form of an aerosol spray from pressuredcontainer or dispenser, which contains a suitable propellant, e.g., agas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

More examples of pharmaceutically acceptable excipients, dosage forms,kits, routes of administration, and methods of treatment can be found inWO 2015051173 and WO 2015051169, the contents of each of which areherein incorporated by reference in their entireties.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the present invention areapparent from the different examples. The provided examples illustratedifferent components and methodology useful in practicing the presentinvention. The examples do not limit the claimed invention. Based on thepresent disclosure the skilled artisan can identify and employ othercomponents and methodology useful for practicing the present invention.

In the synthetic schemes described herein, compounds may be drawn withone particular configuration for simplicity. Such particularconfigurations are not to be construed as limiting the invention to oneor another isomer, tautomer, regioisomer or stereoisomer, nor does itexclude mixtures of isomers, tautomers, regioisomers or stereoisomers;however, it will be understood that a given isomer, tautomer,regioisomer or stereoisomer may have a higher level of activity thananother isomer, tautomer, regioisomer or stereoisomer.

Compounds (including cap analogs) and polynucleotides disclosed herein,or designed, selected and/or optimized by methods described above, onceproduced, can be characterized using a variety of assays known to thoseskilled in the art to determine whether the compounds have biologicalactivity. For example, the molecules can be characterized byconventional assays, including but not limited to protein productionassays (e.g., cell-free translation assays or cell based expressionassays), degradation assays, cell culture assays (e.g., of neoplasticcells), animal models (e.g., rats, mice, rabbits, dogs, or pigs), andthose assays described below, to determine whether they have a predictedactivity, e.g., binding activity and/or binding specificity, andstability.

Furthermore, high-throughput screening can be used to speed up analysisusing such assays. As a result, it can be possible to rapidly screen themolecules described herein for activity, using techniques known in theart. General methodologies for performing high-throughput screening aredescribed, for example, in Devlin (1998) High Throughput Screening,Marcel Dekker; and U.S. Pat. No. 5,763,263. High-throughput assays canuse one or more different assay techniques including, but not limitedto, those described below.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

Example 1: Syntheses of Compounds of the Disclosure Synthesis ofCompound 1

Step 1: Synthesis of Bis-Phosphate Ester (5-2)

To a solution of 5-1 (1.0 g, 0.94 mmol) and ethylene glycol (0.0263 mL,0.47 mmol) in acetonitrile (20 mL) was added 1H-tetrazole inacetonitrile (0.45 M solution, 3.14 mL, 1.41 mmol) dropwise over 3minutes. After stirring at 20° C. for 1.5 h, the reaction mixture wascooled to <−20° C. and treated with t-butylhydroperoxide in n-decane(5.5 M solution, 0.514 mL, 2.83 mmol) over 5 min. The reaction mixturewas allowed to warm to 20° C. overnight. The reaction was quenched withH₂O (Milli Q grade, 60 mL) followed by dichloromethane (60 mL). Theaqueous layer was separated from the organic layer and extracted withdichloromethane (60 mL×2). The combined organic layers were dried oversodium sulfate, filtered through a sintered glass funnel andconcentrated in vacuo at 30° C. to give a pale yellow oil (1.8 g). Theproduct was purified by column chromatography (25 g silica gel) elutinggradient with dichloromethane to 8% methanol in dichloromethane. Theproduct-containing fractions were combined and concentrated in vacuo at30° C. to give the title compound as an off-white solid (449 mg, 47%yield).

¹H NMR (400 MHz, DMSO-d6) −0.49 (s, 6H, 2 CH₃—Si), 0.03 (s, 6H, 2CH₃—Si), 0.79 (s, 18H, 2 tBu-Si), 1.14 (s, 12H, 2 Me₂CH), 2.71 (m, 2H, 2CHMe₂), 2.81 (m, 4H, 2 CH₂CN), 3.17 (m, 2H, 2H-3′), 3.61 (m, 4H,2H₂-5′), 3.70 & 3.73 (2s, 12H, 4OCH₃), 3.91 (m, 2H, 2H-2′), 3.99 (m, 4H,2OCH₂CH₂CN), 4.82 (s, 4H, 2OCH₂Ar), 4.85 (m, 2H, 2H-4′), 6.06 (d, 2H,2H-1′), 6.87-8.21 (m, 34H, 8 Ar), 11.56 (br s, 2H, 2 NH-1), 11.81 (s,2H, 2H-8); ³¹P NMR (161 MHz, D₂O) δ 1.01.

Step 2: Synthesis of Compound 1

A solution of 5-2 (0.31 g, 0.154 mmol) and methanolic ammonia (2 Msolution, 5 mL, 10.0 mmol) was stirred at 20° C. for 4 h andconcentrated in vacuo at 20° C. to give an oil. The oil was dissolved inacetonitrile (6 mL) and N,N-dimethylformamide (3 mL), and treated withtriethylamine trihydrofluoride (0.064 mL, 0.391 mmol) at 20° C. After 3h, triethylamine trihydrofluoride (0.192 mL, 1.173 mmol) was added tothe reaction mixture at 20° C. and the mixture was stirred at 20° C. for3 days. To the reaction mixture was added trifluoroacetic acid (0.015mL, 0.195 mmol) and 1-dodecanethiol (0.103 mL, 0.409 mmol) at 20° C.over 8 minutes. The reaction mixture was stirred at 20° C. for 2 days.1-dodecanethiol (0.052 mL) followed by trifluoroacetic acid (0.345 mL)was added to the reaction mixture, and the mixture was stirredovernight. After 1 day, the reaction was quenched with H₂O (Milli Qgrade, 15 mL) and dichloromethane (10 mL). The aqueous layer wasseparated from the organic layer and extracted with dichloromethane (10mL). The combined organic layers were purified by column chromatography(50 g C18 column) eluting with 10 mM N,N-dimethylhexylammoniumbicarbonate buffer (pH 7.5) to 30% acetonitrile in 10 mMN,N-dimethylhexylammonium bicarbonate buffer (pH 7.5). Theproduct-containing fractions were combined and concentrated in vacuo togive the title compound (197 mg).

¹H NMR (400 MHz, D₂O) δ 0.84 (s, 9H, 3 Me(CH₂)₅N), 1.29 (s) 1.29 (m,18H, 3 MeCH₂CH₂CH₂CH₂CH₂N), 1.67 (m, 6H, 3 CH₂CH₂N), 2.84 (s, 18H, 3Me₂N), 3.09 (m, 6H, 3 NCH₂), 3.98-4.18 (m, 4H, 2H₂-5), 4.24 (m, 2H,2H-2′), 4.43 (m, 2H, 2H-3′), 4.71 (m, 2H, 2H-2′), 5.80 (d, 2H, 2H-1′),7.97 (s, 2H, 2H-8); ³¹P NMR (161 MHz, D₂O) δ 1.03.

Synthesis of Compound 2 Step 1

To a flame dried round bottom flask containing 4 Å molecular sieves inacetonitrile (4 ml) was added2′-tBDSilyl-3′-DMT-Guanosine(n-IPr-PAC)-5′-CED phosphoramidite (0.3 g,0.28 mmol), followed by diethylene glycol (0.03 ml, 0.31 mmol).1H-tetrazole (0.45M in acetonitrile, 0.14 ml, 0.06 mmol) was then addedand the resulting reaction mixture was stirred at ambient temperatureunder N₂ until ³¹P NMR indicated the disappearance of phosphoramidite (3days). Tert-butylhydroperoxide (5.5M in decane, 0.11 ml, 0.6 mmol) wasadded and the resulting reaction mixture was stirred overnight atambient temperature under N₂. The reaction was then filtered andconcentrated to provide crude product which was used without furtherpurification. ³¹P NMR (CD₃CN) δ 139.9 (1P), 140.3 (1P).

Step 2

To a suspension containing the product from Step 1 (0.28 mmol) in THF (5ml) was added methylamine (2M in THF, 1.4 ml, 2.8 mmol). The resultingreaction mixture was stirred at ambient temperature under N2 until ³¹PNMR indicated consumption of starting material and LCMS indicatedremoval of n-isopropyl-PAC protecting group (24 hours). The reaction wasdiluted with water and extracted with dichloromethane. The organics wereconcentrated to provide crude product which was used without furtherpurification. ³¹P NMR (CD₃CN) 8-1.22 (2P).

Step 3

To a solution containing the product from Step 2 (0.28 mmol) in THF (4ml) was added tetrabutylammonium fluoride (1M in THF, 3 ml, 3 mmol). Theresulting reaction mixture was stirred at ambient temperature under N₂until LCMS indicated removal of the 2′ silyl protecting group (16hours). The reaction was diluted with water and extracted withchloroform. The organics were concentrated to provide crude productwhich was used without further purification.

Step 4

To a solution containing the product from Step 3 (0.28 mmol) in THF (3ml) was added trifluoroacetic acid (0.35 ml, 4.5 mmol) followed by1-decanethiol (0.22 ml, 0.9 mmol). The resulting reaction mixture wasstirred at ambient temperature overnight then concentrated under reducedpressure. The resulting crude material was purified by weak anionexchange column chromatography (Sepharose, 0-100% 1M triethylammoniumbicarbonate/water) to provide 0.117 g of the desired product as a whitesolid.

Step 5

The product obtained in Step 4 (0.117 g, 0.11 mmol) was dissolved inwater and the pH was adjusted to 4 by addition of glacial acetic acid.Dimethyl sulfate (0.16 ml, 1.7 mmol) was added dropwise over 90 minutesand pH was maintained between 4.0-4.1 by addition of 5M NaOH. Thereaction was stirred an additional 30 minutes following addition thendiluted with water to 900 ml. The product was purified by weak anionexchange column chromatography (Sepharose, 0-100% 1M triethylammoniumbicarbonate/water) to provide the product as the triethylammonium salt.The triethylammonium salt was then converted to thedimethylhexylammonium salt by reverse phase chromatography (Isco, C18,0-40% 10 mM dimethylhexylammonium bicarbonate/acetonitrile). Lastly, theproduct was converted to the ammonium salt by precipitation withammonium perchlorate/acetone. ¹H NMR (D₂O) δ 3.67 (4H, s), 3.94 (4H,bs), 4.03 (6H, s), 4.15 (2H, m), 4.30 (2H, bs), 4.38 (2H, m), 4.57 (2H,m), 5.94 (2H, m). ³¹P NMR (D₂O) δ 0.32 (2P, s).

Compounds 25 was synthesized in a manner similar to that described abovefor Compound 2.

Compound 25

Synthesis of Compound 7

Step 1

Synthesis of(2R,3R,4R,5R)-2-((((2-((bis(2-cyanoethoxy)phosphoryl)oxy)-3-hydroxypropoxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3,4-diylbis(2-methylpropanoate) (7C).

A 250 mL single-neck round-bottom flask equipped with a stir bar andnitrogen inlet adapter was charged with(2R,3R,4R,5R)-2-((((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3,4-diylbis(2-methylpropanoate) (7A) [3.37 g, 4.86 mmol, 1 eq.] in 27 mL ofCH₃CN (K_(f)=2743 ppm). 3 Å molecular sieves were added to the flask.1-((tert-butyldimethylsilyl)oxy)-3-hydroxypropan-2-ylbis(2-cyanoethyl)phosphate (7B) [1.91 g, 4.86 mmol, 1 eq.] was azeotroped twice withCH₃CN, dissolved in 30 mL of CH₃CN, and added to the reaction flask togive a final K_(f) reading of 1507 ppm. The flask was charged with1H-tetrazole [11.87 mL 0.5 M, 5.34 mmol, 1.1 eq.], resulting in acloudy, white mixture after 5 min. LCMS indicated complete consumptionof the starting guanosine analog after 45 min, at which point the flaskwas cooled to 0° C. in an ice-water bath and charged with tert-Butylhydroperoxide [1.77 mL 5.5 M, 9.72 mmol, 2 eq.]. The reaction mixturestirred at RT for 15 h, and LCMS showed consumption of the intermediateafter 15 h. Filtration and concentration via rotary evaporation afforded6.1 g of a yellow suspension, which was purified through columnchromatography on silica gel (80 g) with 5% MeOH/DCM. Concentration ofthe product-containing fractions yielded 3.7 g (76%) of protectedintermediate,(2R,3R,4R,5R)-5-{[(2-{[bis(2-cyanoethoxy)phosphoryl]oxy}-3-[(tert-butyldimethylsilyl)oxy]propoxy(2-cyanoethoxy)phosphoryl)oxy]methyl}-2-[2-(2-methylpropanamido)-6-oxo-1H-purin-9-yl]-4-[(2-methylpropanoyl)oxy]oxolan-3-yl2-methylpropanoate, as a viscous, colorless oil. A 500 mL single-neckround-bottom flask equipped with a stir bar and nitrogen inlet adapterwas charged with(2R,3R,4R,5R)-5-{[(2-{[bis(2-cyanoethoxy)phosphoryl]oxy}-3-[(tert-butyldimethylsilyl)oxy]propoxy(2-cyanoethoxy)phosphoryl)oxy]methyl}-2-[2-(2-methylpropanamido)-6-oxo-1H-purin-9-yl]-4-[(2-methylpropanoyl)oxy]oxolan-3-yl2-methylpropanoate [3.6 g, 3.6 mmol, 1 eq.] and 100 mL of DCM. The flaskwas then charged with BF₃ etherate [0.89 mL, 7.19 mmol, 2 eq.],immediately causing the colorless solution to turn orange. LC/MS showedlittle consumption of the starting material after 6 min, so anotherequiv. of BF₃ etherate was added. An additional equiv. (total of 4.0equiv.) was added after a total 50 min of reaction time. LC/MS indicatedcomplete consumption of the starting material after 80 min, at whichpoint the reaction mixture was neutralized with 100 mL of 5%NaHCO_(3(aq)) and stirred for 5 min. The aqueous and organic layers inthe cloudy, light orange mixture were separated by using a 500 mLseparatory funnel. The aqueous layer was back-extracted with anadditional 100 mL of DCM. The combined organic layers were concentratedvia rotary evaporation and purified through column chromatography onsilica gel (80 g) with 0-10% MeOH/DCM affording 1.15 g of(2R,3R,4R,5R)-2-((((2-((bis(2-cyanoethoxy)phosphoryl)oxy)-3-hydroxypropoxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3,4-diylbis(2-methylpropanoate) (7C) in 36% yield. ³¹P NMR (D₂O) 8-2.1 (1P),8-2.3 (1P); MS (m/z) 885 [M-H].

Step 2: Synthesis of Compound 7

A 250 mL single-neck round-bottom flask equipped with a stir bar andnitrogen inlet adapter was charged with(2R,3R,4R,5R)-2-({[(2-cyanoethoxy)(diisopropylamino)phosphanyl]oxy}methyl)-5-[2-(2-methylpropanamido)-6-oxo-1H-purin-9-yl]-4-[(2-methylpropanoyl)oxy]oxolan-3-yl2-methylpropanoate (7A)[1.64 g, 2.36 mmol, 1.8 eq.] in 12 mL of MeCN andstirred over 4 Å molecular sieves over about 48 h (K_(f)<1000 ppm).(2R,3R,4R,5R)-5-{[(2-{[bis(2-cyanoethoxy)phosphoryl]oxy}-3-hydroxypropoxy(2-cyanoethoxy)phosphoryl)oxy]methyl}-2-[2-(2-methylpropanamido)-6-oxo-1H-purin-9-yl]-4-[(2-methylpropanoyl)oxy]oxolan-3-yl2-methylpropanoate(7C) [1.15 g, 1.3 mmol, 1 eq.] were dissolved in 4 mLof MeCN and added to the reaction flask to give a final K_(f) reading of<800 ppm. The flask was charged with 1H-tetrazole [2.88 mL 0.5 M, 1.3mmol, 1 eq.], resulting in a cloudy white mixture after 5 min. LCMSindicated complete consumption of the starting guanosine analog after 45min, at which point the flask was cooled to 0° C. in an ice-water bathand charged with tert-Butyl hydroperoxide [0.47 mL 5.5 M, 2.59 mmol, 2eq.]. The reaction mixture stirred at RT overnight, and LCMS showedconsumption of the intermediate by 15 h. Filtration and concentrationvia rotary evaporation afforded a yellow suspension, which was purifiedthrough column chromatography with 5% MeOH/DCM, affording 520 mg of theprotected intermediate (i.e., the intermediate carrying all protectinggroups) in 27% yield.

A 100 mL single-neck round-bottom flask equipped with a stir bar andnitrogen inlet adapter was charged with 0.52 g of the starting material,9 mL of MeCN, and 1,8-Diazabicyclo[5.4.0]undec-7-ene [0.78 mL, 5.22mmol, 15 eq.]. After 1 hour the light brown reaction mixture wasconcentrated to dryness and taken up in 8.5 mL of water. The solutionwas added to methylamine [2.61 mL 2 M, 5.22 mmol, 15 eq.] and 2.6 mL ofammonium hydroxide, and stirred at 60° C. for 2 hours, then cooled toroom temperature and loaded onto a C18 column eluting with DMHAbuffer/CAN. The desired fractions were partially concentrated andlyophilized overnight, affording 150 mg of the fully deprotectedintermediate,[1,3-bis({[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy(hydroxy)phosphoryl}oxy)propan-2-yl]oxyphosphonicacid (7D), in 50% yield.

A 250 mL single-neck round-bottom flask was charged with[1,3-bis({[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy(hydroxy)phosphoryl}oxy)propan-2-yl]oxyphosphonicacid [0.15 g, 0.17 mmol, 1 eq.] and 20 mL of water. The solution isadjusted to pH=4.0 with AcOH. Dimethyl sulfate [1.25 mL, 13.04 mmol, 75eq.] was added in 5 uL portions over 2 hours via syringe pump whilekeeping the pH at 4.0 with 7 uL additions of 5 M NaOH_((aq)). LCMSindicated complete dimethylation. The reaction mixture was diluted with500 mL of water and extracted twice with 400 mL of DCM. The aqueousphase was adjusted to pH=7.8 to match the 1 M triethyl ammoniumbicarbonate buffer. The crude mixture was pumped onto a Sepharosecolumn. The product-containing fractions were combined, mixed with 60 mLof 100 mM DMHA buffer, and pumped onto a 150 g C18 column. The desiredfraction was partially concentrated and then lyophilized overnight. 130mg of the dimethylated product (Compound 7) were obtained in 83% yield.³¹P NMR (D₂O) δ 0.9 (1P), δ 0.1 (1P), 8-0.8 (1P); MS (m/z) 891.2 [M-H]⁻.

Synthesis of Compound 3

Step 1

To a suspension containing guanosine (10.0 g, 35.3 mmol) in acetonitrile(100 ml) was added sodium sulfate (12.5 g, 88.3 mmol) followed byphenylboronic acid (4.52 g, 37.0 mmol). The resulting reaction mixturewas heated to reflux and stirred under N₂ until NMR indicated thecomplete conversion of guanosine (3 hours). The reaction mixture wascooled to ambient temperature and the product was isolated by filtrationto give 11.1 g of a white solid, used without further purification.

Step 2

To a solution containing thiodiethanol (0.75 g, 6.1 mmol) indichloromethane (60 ml) was added diisopropylethylamine (3.2 ml, 18.3mmol) and the reaction was cooled to 0° C. 2-cyanoethylN,N-diisopropylchlorophosphoramidite (2.8 ml, 12.8 mmol) was addeddropwise over 15 minutes. The resulting reaction mixture was allowed towarm to ambient temperature and stirred under N₂. After 2 hours, thereaction was diluted with water and extracted with dichloromethane. Theorganics were washed with water and brine, dried over sodium sulfate andconcentrated. The product was used without further purification.

Step 3

To a solution containing the product from Step 1 (0.71 g, 1.92 mmol) andStep 2 (0.5 g, 0.96 mmol) in DMF (15 ml) was added5-(ethylthio)-1H-tetrazole (0.1 g, 0.72 mmol). The resulting reactionmixture was stirred at ambient temperature under N₂ until ³¹P NMRindicated conversion to desired product (3 hours). The reaction mixturewas concentrated under reduced pressure and used without furtherpurification.

Step 4

To a solution containing the product from Step 3 (1.92 mmol) in THF (20ml) was added tert-butylhydroperoxide (0.7 ml, 3.84 mmol). The resultingreaction mixture was stirred at ambient temperature for 16 hours. DBU(2.9 ml, 19.2 mmol) was added and the reaction was stirred for further16 hours. The reaction mixture was concentrated under reduced pressureand taken up in 900 ml of water. The product was purified by weak anionexchange chromatography (Sepharose, 0-100% 1M triethylammoniumbicarbonate/water).

Step 5

This compound was prepared in a manner similar to Step 5 of synthesizingCompound 2.

Compounds 26-29 were synthesized in a manner similar to that describedabove for Compound 3.

Compounds 26

Compounds 27

Compounds 28

Compounds 29

Example 2: Synthesis of mRNAs by In Vitro Transcription (IVT)

The target mRNAs are prepared following IVT ReactionProtocol—Cotranscriptional capping described herein.

Materials:

Component Stock Conc. Final Conc. Units Desired NTPs 100 Varied mM Cap100 Varied mM 10x Buffer 10 1 X PPIase 0.1 .001 U/uL T7 RNA Polymerase50 14 U/uL Linearized hEPO DNA Varied 100 ng/uL H₂O

-   -   1. Ratio of A:U:C:G varies between 1:1:1:0.1 and 1:1:1:1, with        the cap added in 10-fold excess to G.    -   2. T7 RNA polymerase is added after other components except for        water.    -   3. Water is added for a total reaction volume of 100 uL.    -   4. The mixture is mixed well and spun down in a benchtop        centrifuge for 1 minute.    -   5. The cocktail is incubated at 37 degrees for 4 hours.    -   6. 2.5 uL of RNase free DNase I is added.    -   7. The cocktail is incubated at 37° C. for 45 minutes.

As described in this Example, each of A, U, C, and G includes bothunmodified and modified NTP. After the IVT reaction is complete, themixture is cleaned using membrane purification (MegaClear orequivalent), and Oligo dT. Sample concentration is determined using aspectrophotometer, and degradation is quantitated using a bioanalyzer.

Example 3: Binding Affinities to eIF4E Using Surface Plasmon Resonance(SPR)

General Outline of the Assay Procedure

A sensor chip SA (GE Healthcare) is docked into a Biacore 3000instrument. After washing the surface, protein eIF4E (ElongationInitiation Factor 4E, HNAVIpeptTEVeIF4E 32-217(Biotinylated);pbCPSS1560) is captured non-covalently to the already immobilizedstreptavidin proteins.

Compound concentration series are injected over the immobilized proteinserially in increasing concentration. Interaction models are fittedglobally to the experimental traces, enabling determination of K_(d) orK_(D) (binding affinity; unit: M) and possibly k_(on) (on-rate,calculated from the association phase; unit: M⁻¹ s⁻¹) and k_(off)(off-rate, calculated from the dissociation phase; unit: s⁻¹).

Methods

Preparation of Sensor Chip

A sensor chip (SAD5001 or SA) was docked into a Biacore 3000 instrument,washed with 50 mM NaOH, 1M NaCl. Protein eIF4E was diluted in runningbuffer (50 mM HEPES, 150 mM KCl, 10 mM MgCl₂, 2 mM TCEP) to ˜1 μM. Thediluted protein solution was injected for 300-600 seconds. Typicalcapture levels were 5000-6000 RU.

Test compounds were solubilized in ddH₂O or DMSO to 10 mM. 100 μM stockswere prepared by 100-fold dilution in running buffer (50 mM HEPES, 150mM KCl, 10 mM MgCl₂). Assay was run with or without 1% DMSO.

Data were analyzed in GeneData. Curve fit was accepted or rejected bylooking at the resulting sensorgrams and steady state fits.

Assay Validation

eIF4E protein was captured according to the above procedure and a set of7-methyl (m7) guanosine phosphate compounds (m7GMP, m7GDP, m7GTP) aswell as a compound with an extra guanosine residue after the triphosphate chain (m7GTPG) were injected in dose response. Assay has beenvalidated using running buffer with and without DMSO. It was found thatsurface activity and K_(d) for m7GTP is not affected by DMSO. It wasalso found that the surface is extremely stable (continuous use for >6weeks resulted in 5-10% loss of surface activity). Further, newlycaptured protein stabilizes slowly, leading to negative responses duringthe dissociation phase for compounds injected over newly capturedprotein.

Table 3 below includes the results for certain compounds of thedisclosure.

TABLE 3 Compound No. K_(d) (μM) k_(off) (s⁻¹) τ (s) Cap0 (i.e., m7GpppG)2 0.8  1.25 2 110 TBD 7 6.5 0.067 15 25 300 TBD 39 45 TBD

Example 4: Kinetic Cell Free In Vitro Translation Assay and CapCompetition Assay

The in vitro translation assay was conducted with the HeLa 1-stepcoupled IVT kit (ThermoFisher Scientific, Waltham, Mass.) according tothe manufacturer's instructions to assess performance of new cap analogsas free compounds or as an integral part of capped mRNA. Cap analogswith affinity to eIF4E protein may reduce protein synthesis rate incell-free translation. Further, RNAs containing such cap analogs(“Cap-modRNA”) show different potency of protein synthesis in cell-freetranslation.

The modified RNAs (“modRNAs”) of eGFP and mCitrine-degron, harboringchemical modifications on either the CAP structures, selected riboseunits and/or the bases, were diluted in sterile nuclease-free water to afinal amount of 500 ng in 5 uL. This volume was added to 20 uL offreshly prepared HeLa Lysate. The in vitro translation reaction was donein a standard 96-well round bottom plate (Corning, Corning, N.Y.),covered with an self-adhesive fluorescence-compatible seal (BioRad,Hercules, Calif.) at 30° C. inside the plate reader Cytation 3 (BioTek,Winooski, Vt.).

The fluorescent signal per reaction increased over time and isconsidered proportional to the occurring protein synthesis. Eachcell-free translation reaction was monitored for 120-180 min with thefollowing settings: eGFP protein—ex. 485 nm, em. 515 nm, gain 80;mCitrine-degron protein—ex. 515, em. 545, gain 70 or 80. The height ofthe reading head was set to 1 mm above the plate and a reading speed ofone per sample every 17 seconds.

For competition assays, the total volume of the cell-free translationreaction was increased to 27.8 uL by addition of either water or dilutedfree CAP analogs in water. The stock concentration of the free CAPanalogs was 1 mM. With two-fold dilutions in water, the concentrationwas reduced sequentially. After cell-free translation reaction, modRNA(e.g., an m7GpppG(2′-Om) capped mRNA (i.e., a Cap1-tipped mRNA) codingfor eGFP) and diluted CAP analogs were combined, the titration curve hada final concentration of 100 uM, 50 uM, 25 uM, 12.5 uM, 6.25 uM, 3.12 uMand 0 uM of free CAP analogs. The CAP analogs used in this study wereeither commercial products serving as reference material (TriLink, SanDiego, Calif.) or compounds disclosed herein. It is hypothesized thatthe small molecule cap analogs interfere with the assembly of the“closed loop” in a K_(d)-dependent fashion.

After the fluorescent signal in cell-free translation reaction reached astable plateau, absolute values thereof were transferred to astatistical analysis program (GraphPad Software, La Jolla, Calif.) andcurve fitting or IC₅₀ calculations were derived with settings accordingto the instructions of the manufacturer.

The results from the cap competition assay are illustrated in FIG. 1. Inthis study, the modRNA used comprises 1-methyl-pseudouridine, whichreplaces each uridine in the RNA sequence and 5-methyl cytidine, whichreplaces each cytidine in the RNA sequence. Further, Table 4 belowincludes the IC₅₀ values of certain compounds of the disclosure.

TABLE 4 Compound No. IC₅₀ (μM) K_(d) (μM) Cap0 (i.e., m7GpppG) 36 2 7 236.5

Cell free translation assays were also conducted using modRNAs comprises5-methoxy uridine, which replaces each uridine in the RNA sequence,except otherwise specified. The results are shown in Table 5. Table 5discloses the hEPO levels after 3 hours of a cell-free translationassay.

TABLE 5 Compound No. CFT (norm to conc. & capping & cap1) τ (s) 7 1.853.7 Cap1 1.00 1.3 ARCA 1.93 0.6

Example 5: Cell-Based Expression Assay

The cell-based expression assay was conducted following the protocol asdescribed below.

-   -   1) Day 1: Seed Hela/Vero/BJ-Fibroblast at 20K cells in 100 uL        media/well of a 96 well plate    -   2) Day 2: Transfection        -   Transfect 250 ng/rxn on mCherry/deg mCitrine; 25 ng/rxn on            nanoLuc        -   Dilute nanoLuc mRNA to 10 ng/uL, in 96 well plates.        -   Plate map from Manufacturing (100 ng/uL, per well)

1 2 3 4 5 6 7 8 9 10 11 12 mcherry A M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11M12 B M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24 C D E F nanoluc GN1 M2 M3 M4 M5 M6 M7 M8 M9 M10 N11 N12 H N13 N14 N15 N16 N17 N18 N19 N20N21 N22 N23 N24

-   -   -   -   Make a NanoLuc Dilution Plate (1:10 dil from                manufactory, given 10 ng/uL, per well)

        -   Master mix plate map:

1 2 3 4 5 6 7 8 9 10 11 12 A B media LF2000 G0 G1 G2 G5 G0(N21) G1(N22)G1(N23) G5(N24) C D B8.1 B8.2 B8.3 B8.4 B8.5 B8.6 B8.7 B8.8 B8.9 B8.10 EF B8.11 B8.12 B8.13 B8.14 B8.15 B8.16 B8.17 B8.18 B8.19 B8.20 G H

-   -   -   -   Make a mCherry/deg mCitrine Master mix plate and a                nanoLuc Master mix plate for duplicates, using the                layout above.

        -   Stamp out mCherry/deg mCitrine samples directly from            manufactory plate.

        -   Using the same plate map as NanoLuc.

        -   Destination Plate map (Cell plates):

1 2 3 4 5 6 7 8 9 10 11 12 A B media LF2000 G0 G1 G2 G5 G0(N21) G1(N22)G2(N23) G5(N24) C media LF2000 G0 G1 G2 G5 G0(N21) G1(N22) G2(N23)G5(N24) D B8.1 B8.2 B8.3 B8.4 B8.5 B8.6 B8.7 B8.8 B8.9 B8.10 E B8.1 B8.2B8.3 B8.4 B8.5 B8.6 B8.7 B8.8 B8.9 B8.10 F B8.11 B8.12 B8.13 B8.14 B8.15B8.16 B8.17 B8.18 B8.19 B8.20 G B8.11 B8.12 B8.13 B8.14 B8.15 B8.16B8.17 B8.18 B8.19 B8.20 H

1 RNX 4 RNX mRNA 2.5 uL 10 uL Lipo 2K 0.5 uL 2 uL Optimem 17 uL 68 uLTotal 20 uL

-   -   -   -   Incubate Lipofectamine/Optimem for 15 mins, 70 uL added                to each well of master mix plate.            -   Add 10 uL of mRNA (per well) to 70 uL L2K/Optimem                mixture.            -   Incubate mRNA with L2K/Optimem mixture for another 15                mins.            -   Add 20 uL of mRNA mixture to each well of CELL PLATE.

    -   3) Day 3: Assay (24 hours for expression; 48 hours for        cytokine):        -   mCherry:            -   Wash with 100 uL PBS 1×            -   Add 100 uL PBS for reading            -   Take read on Synergy:            -   Program: Fluorescence Endpoint at Excitation: 585,                emission: 615,            -   Gain: 100        -   Degron mCitrine            -   Wash with 100 uL PBS 1×            -   Add 100 uL PBS for reading            -   Take reads on Synergy at Excitation: 510; emission: 540,                Gain: 100.        -   NanoLuc:            -   Wash with 100 uL PBS 1×            -   Add 100 uL Glo Lysis buffer 1×            -   Take reads on Synergy            -   Program: Luminescence at Gain 115 (default)

    -   4) Day 4 Assay (IFN-b ASSAY):        -   Use VeriKine Human Interferon Beta ELISA Kit (#41410-2, PBL            Biosciences)        -   Follow the protocol of the kit.

The results from the cell-based expression assays in human primaryhepatocytes are listed in Table 6. In this study, each of the mRNAscarrying various caps (e.g., Cap1, ARCA or cap analogs disclosed herein)also comprises 5-methoxy uridine, which replaces each uridine in the RNAsequence. Table 6 below shows the normalized expression level usingmodified mRNAs carrying various caps as compared to mRNA carrying Cap1,in which, mRNA carrying Compound 7 is unmethylated at 2′-OH of thepenultimate guanosine (Cap0-like) while all other caps are Cap1-like,i.e., containing the structure of pppG(2′-Om).

TABLE 6 Compound No. h-primHeps norm to Capping and Cap1 7 0.22 Cap11.00 ARCA 1.52

Example 6: In Vivo Expression Assay

mRNAs encoding hEPO are synthesized according to the method described inExample 2 above, co-transcriptionally incorporating cap analogs of thedisclosure. As in the study of Example 5, each of the mRNAs carryingvarious caps (e.g., Cap1, ARCA, or cap analogs disclosed herein) alsocomprises 5-methoxy uridine, which replaces each uridine in the RNAsequence. A MC3-based lipid nanoparticle (LNP) formulation of thesynthesized mRNA is produced, and is intravenously administered to CD-1mice (n=3) at a bolus dose of 0.05 mg/kg. The level of hEPO was testedat 6 h, 24 h, or 48 h after injection. Table 7 shows the normalized hEPOlevels measured at 6 h after injection, in which, mRNA carrying Compound7 is unmethylated at 2′-OH of the penultimate guanosine (Cap0-like)while all other caps are Cap1-like, i.e., containing the structure ofpppG(2′-Om).

TABLE 7 in vivo hEPO normalized to capping Compound No. capping %/100and Cap1 7 0.68 0.17 Cap1 1 1.00 ARCA 0.86 0.70

The invention can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. An RNA molecule having a 5′ end comprising acompound of formula (III):

wherein B₁ is

R_(a), R_(b), and R_(c) are H; R₁ is CH₃; B₂ is

R_(d), R_(e), and R_(f) are H; X₁ is N or N⁺(CH₃); X₂ is O; R₂₀, R₂₁,R₂₂, and R₂₃ are H; R₂₇ and R₂₈ are OH; R₂ is OR₃; R₃ is H or CH₃; andY₂ is

wherein

indicates an attachment point.
 2. The RNA molecule of claim 1, whereinX₁ is N.
 3. The RNA molecule of claim 1, wherein R₃ is CH₃.
 4. The RNAmolecule of claim 1, wherein X₁ is N⁺(CH₃).
 5. The RNA molecule of claim1, wherein R₃ is H.
 6. The RNA molecule of claim 1, wherein R₃ is H andX₁ is N.
 7. The RNA molecule of claim 1, wherein R₃ is H and X₁ isN⁺(CH₃).
 8. The RNA molecule of claim 1, wherein R₃ is CH₃ and X₁ isN⁺(CH₃).
 9. The RNA molecule of claim 1, having a 5′ end comprising

wherein

indicates the attachment point.
 10. The RNA molecule of claim 9, whereinX₁ is N.
 11. The RNA molecule of claim 9, wherein R₃ is CH₃.
 12. The RNAmolecule of claim 9, wherein X₁ is N⁺(CH₃).
 13. The RNA molecule ofclaim 9, wherein R₃ is H.
 14. The RNA molecule of claim 9, wherein R₃ isH and X₁ is N.
 15. The RNA molecule of claim 9, wherein R₃ is H and X₁is N⁺(CH₃).
 16. The RNA molecule of claim 9, wherein R₃ is CH₃ and X₁ isN⁺(CH₃)
 17. The RNA molecule of claim 1, having a 5′ end comprising

wherein

indicates the attachment point.
 18. The RNA molecule of claim 1, whereinthe RNA molecule has a half-life that is at least 1.2 times of that of acorresponding natural RNA molecule in a cellular environment.
 19. TheRNA molecule of claim 9, wherein the RNA molecule has a half-life thatis at least 1.2 times of that of a corresponding natural RNA molecule ina cellular environment.
 20. The RNA molecule of claim 17, wherein theRNA molecule has a half-life that is at least 1.2 times of that of acorresponding natural RNA molecule in a cellular environment.